summary of transcription in protein synthesis

* Methionine was removed in all of these proteins

** Methionine was not removed from any of these proteins

Once the initiation complex is formed on the mRNA, the large ribosomal subunit binds to this complex, which causes the release of IFs (initiation factors). The large subunit of the ribosome has three sites at which tRNA molecules can bind. The A (amino acid) site is the location at which the aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that correct amino acid is added to the growing polypeptide chain. The P (polypeptide) site is the location at which the amino acid is transferred from its tRNA to the growing polypeptide chain. Finally, the E (exit) site is the location at which the "empty" tRNA sits before being released back into the cytoplasm to bind another amino acid and repeat the process. The initiator methionine tRNA is the only aminoacyl-tRNA that can bind in the P site of the ribosome, and the A site is aligned with the second mRNA codon. The ribosome is thus ready to bind the second aminoacyl-tRNA at the A site, which will be joined to the initiator methionine by the first peptide bond (Figure 5).

The Elongation Phase

Next, peptide bonds between the now-adjacent first and second amino acids are formed through a peptidyl transferase activity. For many years, it was thought that an enzyme catalyzed this step, but recent evidence indicates that the transferase activity is a catalytic function of rRNA (Pierce, 2000). After the peptide bond is formed, the ribosome shifts, or translocates, again, thus causing the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to pick up another amino acid. In addition, the A site is now empty and ready to receive the tRNA for the next codon.

This process is repeated until all the codons in the mRNA have been read by tRNA molecules, and the amino acids attached to the tRNAs have been linked together in the growing polypeptide chain in the appropriate order. At this point, translation must be terminated, and the nascent protein must be released from the mRNA and ribosome.

Termination of Translation

There are three termination codons that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA. No tRNAs recognize these codons. Thus, in the place of these tRNAs, one of several proteins, called release factors, binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.

Comparing Eukaryotic and Prokaryotic Translation

The translation process is very similar in prokaryotes and eukaryotes. Although different elongation, initiation, and termination factors are used, the genetic code is generally identical. As previously noted, in bacteria, transcription and translation take place simultaneously, and mRNAs are relatively short-lived. In eukaryotes, however, mRNAs have highly variable half-lives, are subject to modifications, and must exit the nucleus to be translated; these multiple steps offer additional opportunities to regulate levels of protein production, and thereby fine-tune gene expression.

References and Recommended Reading

Chapeville, F., et al. On the role of soluble ribonucleic acid in coding for amino acids. Proceedings of the National Academy of Sciences 48 , 1086–1092 (1962)

Crick, F. On protein synthesis. Symposia of the Society for Experimental Biology 12 , 138–163 (1958)

Flinta, C., et al . Sequence determinants of N-terminal protein processing. European Journal of Biochemistry 154 , 193–196 (1986)

Grunberger, D., et al . Codon recognition by enzymatically mischarged valine transfer ribonucleic acid. Science 166 , 1635–1637 (1969) doi:10.1126/science.166.3913.1635

Kozak, M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo . Nature 308 , 241–246 (1984) doi:10.1038308241a0 ( link to article )

---. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44 , 283–292 (1986)

---. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15 , 8125–8148 (1987)

Pierce, B. A. Genetics: A conceptual approach (New York, Freeman, 2000)

Shine, J., & Dalgarno, L. Determinant of cistron specificity in bacterial ribosomes. Nature 254 , 34–38 (1975) doi:10.1038/254034a0 ( link to article )

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3.4 Protein Synthesis

Learning objectives.

Main Objective

• Explain the process by which a cell builds proteins using the DNA code

By the end of this section, you will be able to:

• Explain how the genetic code within DNA determines the proteins formed
• Describe the process of transcription
• Explain the process of translation
• Discuss the function of ribosomes

It was mentioned earlier that DNA provides a “blueprint” for the cell structure and physiology. This refers to the fact that DNA contains the information necessary for the cell to build one very important type of molecule: the protein. Most structural components of the cell are made up, at least in part, by proteins and virtually all the functions that a cell carries out are completed with the help of proteins. One of the most important classes of proteins is enzymes, which help speed up necessary biochemical reactions that take place inside the cell. Some of these critical biochemical reactions include building larger molecules from smaller components (such as what occurs during DNA replication or synthesis of microtubules) and breaking down larger molecules into smaller components (such as when harvesting chemical energy from nutrient molecules). Whatever the cellular process may be, it is almost sure to involve proteins. Just as the cell’s genome describes its full complement of DNA, a cell’s proteome is its full complement of proteins. Protein synthesis begins with genes. A gene is a functional segment of DNA that provides the genetic information necessary to build a protein. Each particular gene provides the code necessary to construct a particular protein. Gene expression , which transforms the information coded in a gene to a final gene product, ultimately dictates the structure and function of a cell by determining which proteins are made.

The interpretation of genes works in the following way. Recall that proteins are polymers, or chains, of many amino acid building blocks. The sequence of bases in a gene (that is, its sequence of A, T, C, G nucleotides) translates to an amino acid sequence. A triplet is a section of three DNA bases in a row that codes for a specific amino acid. For example, the DNA triplet CAC (cytosine, adenine, and cytosine) specifies the amino acid valine. Therefore, a gene, which is composed of multiple triplets in a unique sequence, provides the code to build an entire protein, with multiple amino acids in the proper sequence ( Figure 3.4.1 ). The mechanism by which cells turn the DNA code into a protein product is a two-step process, with an RNA molecule as the intermediate.

From DNA to RNA: Transcription

DNA is housed within the nucleus, and protein synthesis takes place in the cytoplasm, thus there must be some sort of intermediate messenger that leaves the nucleus and manages protein synthesis. This intermediate messenger is messenger RNA (mRNA) , (Figure 3.29), a single-stranded nucleic acid that carries a copy of the genetic code for a single gene out of the nucleus and into the cytoplasm where it is used to produce proteins.

There are several different types of RNA, each having different functions in the cell. The structure of RNA is similar to DNA with a few small exceptions. For one thing, unlike DNA, most types of RNA, including mRNA, are single-stranded and contain no complementary strand. Second, the ribose sugar in RNA contains an additional oxygen atom compared with DNA. Finally, instead of the base thymine, RNA contains the base uracil. This means that adenine will always pair up with uracil during the protein synthesis process.

Gene expression begins with the process called transcription , which is the synthesis of a strand of mRNA that is complementary to the gene of interest. This process is called transcription because the mRNA is like a transcript, or copy, of the gene’s DNA code. Transcription begins in a fashion somewhat like DNA replication, in that a region of DNA unwinds and the two strands separate, however, only that small portion of the DNA will be split apart. The triplets within the gene on this section of the DNA molecule are used as the template to transcribe the complementary strand of RNA ( Figure 3.4.2 ). A codon is a three-base sequence of mRNA, so-called because they directly encode amino acids. Like DNA replication, there are three stages to transcription: initiation, elongation, and termination.

In the first of the two stages of making protein from DNA, a gene on the DNA molecule is transcribed into a complementary mRNA molecule.

Stage 1: Initiation. A region at the beginning of the gene called a promoter—a particular sequence of nucleotides—triggers the start of transcription.

Stage 2: Elongation. Transcription starts when RNA polymerase unwinds the DNA segment. One strand, referred to as the coding strand, becomes the template with the genes to be coded. The polymerase then aligns the correct nucleic acid (A, C, G, or U) with its complementary base on the coding strand of DNA. RNA polymerase is an enzyme that adds new nucleotides to a growing strand of RNA. This process builds a strand of mRNA.

Stage 3: Termination. When the polymerase has reached the end of the gene, one of three specific triplets (UAA, UAG, or UGA) codes a “stop” signal, which triggers the enzymes to terminate transcription and release the mRNA transcript.

The transcription process is regulated by a class of proteins called transcription factors , which bind to the gene sequence and either promote or inhibit their transcription.   (move Figure 3.35 here).

Before the mRNA molecule leaves the nucleus and proceeds to protein synthesis, it is modified in a number of ways. For this reason, it is often called a pre-mRNA at this stage. For example, your DNA, and thus complementary mRNA, contains long regions called non-coding regions that do not code for amino acids. Their function is still a mystery, but the process called splicing removes these non-coding regions from the pre-mRNA transcript ( Figure 3.4.3 ). A spliceosome —a structure composed of various proteins and other molecules—attaches to the mRNA and “splices” or cuts out the non-coding regions. The removed segment of the transcript is called an intron . The remaining exons are pasted together. An exon is a segment of RNA that remains after splicing. Interestingly, some introns that are removed from mRNA are not always non-coding. When different coding regions of mRNA are spliced out, different variations of the protein will eventually result, with differences in structure and function. This process results in a much larger variety of possible proteins and protein functions. When the mRNA transcript is ready, it travels out of the nucleus and into the cytoplasm.

External Website

This video will show you the important enzymes and biomolecules involved in the process of transcription, the process of making an mRNA molecule from DNA.

From RNA to Protein: Translation

Like translating a book from one language into another, the codons on a strand of mRNA must be translated into the amino acid alphabet of proteins. Translation is the process of synthesizing a chain of amino acids called a polypeptide. Translation requires two major aids: first, a “translator,” the molecule that will conduct the translation, and second, a substrate on which the mRNA strand is translated into a new protein, like the translator’s “desk.” Both of these requirements are fulfilled by other types of RNA. The substrate on which translation takes place is the ribosome.

Remember that many of a cell’s ribosomes are found associated with the rough ER, and carry out the synthesis of proteins destined for the Golgi apparatus. Ribosomal RNA (rRNA) is a type of RNA that, together with proteins, composes the structure of the ribosome. Ribosomes exist in the cytoplasm as two distinct components, a small and a large subunit. When an mRNA molecule is ready to be translated, the two subunits come together and attach to the mRNA. The ribosome provides a substrate for translation, bringing together and aligning the mRNA molecule with the molecular “translators” that must decipher its code.

The other major requirement for protein synthesis is the translator molecules that physically “read” the mRNA codons. Transfer RNA (tRNA) is a type of RNA that ferries the appropriate corresponding amino acids to the ribosome, and attaches each new amino acid to the last, building the polypeptide chain one-by-one. Thus tRNA transfers specific amino acids from the cytoplasm to a growing polypeptide. The tRNA molecules must be able to recognize the codons on mRNA and match them with the correct amino acid. The tRNA is modified for this function. On one end of its structure is a binding site for a specific amino acid. On the other end is a base sequence that matches the codon specifying its particular amino acid. This sequence of three bases on the tRNA molecule is called an anticodon . For example, a tRNA responsible for shuttling the amino acid glycine contains a binding site for glycine on one end. On the other end it contains an anticodon that complements the glycine codon (GGA is a codon for glycine, and so the tRNAs anticodon would read CCU). Equipped with its particular cargo and matching anticodon, a tRNA molecule can read its recognized mRNA codon and bring the corresponding amino acid to the growing chain ( Figure 3.4.4 ).

Much like the processes of DNA replication and transcription, translation consists of three main stages: initiation, elongation, and termination. Initiation takes place with the binding of a ribosome to an mRNA transcript. The elongation stage involves the recognition of a tRNA anticodon with the next mRNA codon in the sequence. Once the anticodon and codon sequences are bound (remember, they are complementary base pairs), the tRNA presents its amino acid cargo and the growing polypeptide strand is attached to this next amino acid. This attachment takes place with the assistance of various enzymes and requires energy. The tRNA molecule then releases the mRNA strand, the mRNA strand shifts one codon over in the ribosome, and the next appropriate tRNA arrives with its matching anticodon. This process continues until the final codon on the mRNA is reached which provides a “stop” message that signals termination of translation and triggers the release of the complete, newly synthesized protein. Thus, a gene within the DNA molecule is transcribed into mRNA, which is then translated into a protein product ( Figure 3.4.5 ).

This video will show you the important enzymes and biomolecules involved in the process of translation, which uses mRNA to code for a protein.

Commonly, an mRNA transcription will be translated simultaneously by several adjacent ribosomes. This increases the efficiency of protein synthesis. A single ribosome might translate an mRNA molecule in approximately one minute; so multiple ribosomes aboard a single transcript could produce multiple times the number of the same protein in the same minute. A polyribosome is a string of ribosomes translating a single mRNA strand.

Watch this  video  to learn about ribosomes. The ribosome binds to the mRNA molecule to start translation of its code into a protein. What happens to the small and large ribosomal subunits at the end of translation?

Chapter Review

DNA stores the information necessary for instructing the cell to perform all of its functions. Cells use the genetic code stored within DNA to build proteins, which ultimately determines the structure and function of the cell. This genetic code lies in the particular sequence of nucleotides that make up each gene along the DNA molecule. To “read” this code, the cell must perform two sequential steps. In the first step, transcription, the DNA code is converted into a RNA code. A molecule of messenger RNA that is complementary to a specific gene is synthesized in a process similar to DNA replication. The molecule of mRNA provides the code to synthesize a protein. In the process of translation, the mRNA attaches to a ribosome. Next, tRNA molecules shuttle the appropriate amino acids to the ribosome, one-by-one, coded by sequential triplet codons on the mRNA, until the protein is fully synthesized. When completed, the mRNA detaches from the ribosome, and the protein is released. Typically, multiple ribosomes attach to a single mRNA molecule at once such that multiple proteins can be manufactured from the mRNA concurrently.

Review Questions

Critical thinking questions.

Briefly explain the similarities between transcription and DNA replication.

Transcription and DNA replication both involve the synthesis of nucleic acids. These processes share many common features—particularly, the similar processes of initiation, elongation, and termination. In both cases the DNA molecule must be untwisted and separated, and the coding (i.e., sense) strand will be used as a template. Also, polymerases serve to add nucleotides to the growing DNA or mRNA strand. Both processes are signaled to terminate when completed.

Contrast transcription and translation. Name at least three differences between the two processes.

Transcription is really a “copy” process and translation is really an “interpretation” process, because transcription involves copying the DNA message into a very similar RNA message whereas translation involves converting the RNA message into the very different amino acid message. The two processes also differ in their location: transcription occurs in the nucleus and translation in the cytoplasm. The mechanisms by which the two processes are performed are also completely different: transcription utilizes polymerase enzymes to build mRNA whereas translation utilizes different kinds of RNA to build protein.

This work, Anatomy & Physiology, is adapted from Anatomy & Physiology by OpenStax , licensed under CC BY . This edition, with revised content and artwork, is licensed under CC BY-SA except where otherwise noted.

Images, from Anatomy & Physiology by OpenStax , are licensed under CC BY except where otherwise noted.

Access the original for free at https://openstax.org/books/anatomy-and-physiology/pages/1-introduction .

Anatomy & Physiology Copyright © 2019 by Lindsay M. Biga, Staci Bronson, Sierra Dawson, Amy Harwell, Robin Hopkins, Joel Kaufmann, Mike LeMaster, Philip Matern, Katie Morrison-Graham, Kristen Oja, Devon Quick, Jon Runyeon, OSU OERU, and OpenStax is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License , except where otherwise noted.

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transcription , the synthesis of RNA from DNA . Genetic information flows from DNA into protein , the substance that gives an organism its form. This flow of information occurs through the sequential processes of transcription (DNA to RNA) and translation (RNA to protein). Transcription occurs when there is a need for a particular gene product at a specific time or in a specific tissue.

During transcription, only one strand of DNA is usually copied. This is called the template strand, and the RNA molecules produced are single-stranded messenger RNAs (mRNAs). The DNA strand that would correspond to the mRNA is called the coding or sense strand. In eukaryotes (organisms that possess a nucleus ) the initial product of transcription is called a pre-mRNA. Pre-mRNA is extensively edited through splicing before the mature mRNA is produced and ready for translation by the ribosome , the cellular organelle that serves as the site of protein synthesis. Transcription of any one gene takes place at the chromosomal location of that gene, which is a relatively short segment of the chromosome . The active transcription of a gene depends on the need for the activity of that particular gene in a specific cell or tissue or at a given time.

Small segments of DNA are transcribed into RNA by the enzyme RNA polymerase , which achieves this copying in a strictly controlled process. The first step is to recognize a specific sequence on DNA called a promoter that signifies the start of the gene. The two strands of DNA become separated at this point, and RNA polymerase begins copying from a specific point on one strand of the DNA using a special type of sugar -containing nucleoside called ribonucleoside 5’-triphosphate to begin the growing chain. Additional ribonucleoside triphosphates are used as the substrate, and, by cleavage of their high-energy phosphate bond, ribonucleoside monophosphates are incorporated into the growing RNA chain. Each successive ribonucleotide is directed by the complementary base pairing rules of DNA. For example, a C ( cytosine ) in DNA directs the incorporation of a G ( guanine ) into RNA. Likewise, a G in DNA is copied into a C in RNA, a T ( thymine ) into an A ( adenine ), and an A into a U ( uracil ; RNA contains U in place of the T of DNA). Synthesis continues until a termination signal is reached, at which point the RNA polymerase drops off the DNA, and the RNA molecule is released.

Ahead of many genes in prokaryotes (organisms that lack a nucleus), there are signals called “operators” ( see operons ) where specialized proteins called repressors bind to the DNA just upstream of the start point of transcription and prevent access to the DNA by RNA polymerase. These repressor proteins thus prevent transcription of the gene by physically blocking the action of the RNA polymerase. Typically, repressors are released from their blocking action when they receive signals from other molecules in the cell indicating that the gene needs to be expressed. Ahead of some prokaryotic genes are signals to which activator proteins bind to stimulate transcription.

Transcription in eukaryotes is more complicated than in prokaryotes. First, the RNA polymerase of higher organisms is a more complicated enzyme than the relatively simple five-subunit enzyme of prokaryotes. In addition, there are many more accessory factors that help to control the efficiency of the individual promoters. These accessory proteins are called transcription factors and typically respond to signals from within the cell that indicate whether transcription is required. In many human genes, several transcription factors may be needed before transcription can proceed efficiently. A transcription factor can cause either repression or activation of gene expression in eukaryotes.

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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

Chapter 7 protein synthesis, processing, and regulation.

Transcription and RNA processing are followed by translation, the synthesis of proteins as directed by mRNA templates. Proteins are the active players in most cell processes, implementing the myriad tasks that are directed by the information encoded in genomic DNA. Protein synthesis is thus the final stage of gene expression. However, the translation of mRNA is only the first step in the formation of a functional protein. The polypeptide chain must then fold into the appropriate three-dimensional conformation and, frequently, undergo various processing steps before being converted to its active form. These processing steps, particularly in eukaryotes, are intimately related to the sorting and transport of different proteins to their appropriate destinations within the cell.

Although the expression of most genes is regulated primarily at the level of transcription (see Chapter 6), gene expression can also be controlled at the level of translation, and this control is an important element of gene regulation in both prokaryotic and eukaryotic cells. Of even broader significance, however, are the mechanisms that control the activities of proteins within cells. Once synthesized, most proteins can be regulated in response to extracellular signals by either covalent modifications or by association with other molecules. In addition, the levels of proteins within cells can be controlled by differential rates of protein degradation. These multiple controls of both the amounts and activities of intracellular proteins ultimately regulate all aspects of cell behavior.

• Translation of mRNA
• Protein Folding and Processing
• Regulation of Protein Function
• Protein Degradation
• References and Further Reading
• Cite this Page Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Chapter 7, Protein Synthesis, Processing, and Regulation.

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16 Protein Synthesis Overview

Andrea Bierema

Learning Objectives

Students will be able to:

• Describe the structure and purpose of DNA and RNA.
• Describe the general process of protein synthesis.
• Describe the molecular anatomy of genes and genomes.
• Identify DNA and mRNA bases and binding patterns.
• Interpret a codon-amino acid chart.
• Given a DNA sequence, determine the corresponding mRNA sequence and amino acid sequence.

Central Dogma

The central dogma of molecular biology is that DNA codes for RNA and RNA codes for protein. In addition to DNA coding for RNA, much of the DNA regulates the synthesis of RNA- which ultimately means that it regulates the synthesis of protein. We will learn about protein synthesis regulation in a later chapter.

Protein synthesis consists of two main processes: transcription and translation. During the process of transcription —which occurs in the nucleus—an mRNA molecule is created by reading the DNA. Note that DNA never “becomes” RNA; rather, the DNA is “read” to make an RNA molecule. The mRNA leaves the nucleus and then, through the process of translation , the mRNA is read to create an amino acid sequence which folds into a protein.

Consider what the terms “transcribe” and “translate” mean in relation to language. To “transcribe” something means to rewrite text again in the same language while to “translate” something means to rewrite the text in a different language. Similar to these meanings, in biology, DNA is transcribed into RNA: both DNA and RNA are made of nucleic acid (i.e., the same “language”). With the assistance of proteins, DNA is “read” and transcribed into an mRNA sequence. To read RNA and create protein, though, we refer to it as being translated: RNA is made of nucleic acid and protein is made of amino acid (i.e., different “languages”). Therefore, DNA is transcribed to create an mRNA sequence, and then the mRNA sequence is translated to make a protein.

DNA and RNA

The two main types of nucleic acids are  deoxyribonucleic acid (DNA)  and  ribonucleic acid (RNA) . DNA is the genetic material in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.

The cell’s entire genetic content is its genome, and the study of genomes is genomics. In eukaryotic cells but not in prokaryotes, a DNA molecule may contain tens of thousands of genes. Many genes contain information to make protein products (e.g., mRNA). Other genes code for RNA products. DNA controls all of the cellular activities by turning the genes “on” or “off.”

The other type of nucleic acid, RNA, is mostly involved in protein synthesis. The DNA molecules never leave the nucleus but instead use an intermediary to communicate with the rest of the cell. This intermediary is the  messenger RNA (mRNA) . Other types of RNA—like rRNA, tRNA, and microRNA—are involved in protein synthesis and its regulation.

DNA and RNA are comprised of monomers that scientists call  nucleotides . The nucleotides combine with each other to form a  polynucleotide , DNA or RNA. Three components comprise each nucleotide: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group. Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups. Therefore, although the terms “base” and “nucleotide” are sometimes used interchangeably, a nucleotide contains a base as well as part of the sugar-phosphate backbone.

Comparison of RNA (left molecule) and DNA (right molecule). The color of the bases in RNA and DNA aligns with the colored boxes next to each base molecule.

Examine the image above and then answer the following questions:

What is a Gene?

The gene is the basic physical unit of inheritance. Genes are passed from parents to offspring and contain the information needed to specify traits. Genes are arranged, one after another, on structures called chromosomes. A chromosome contains a single, long DNA molecule, only a portion of which corresponds to a single gene. Humans have approximately 20,000 genes arranged on their chromosomes. Watch the following video for an animated view on the relationship between chromosomes and genes.

Protein Synthesis Overview

The two main processes in protein synthesis are transcription and translation. The following is an overview of each of these processes. Each process will be described in more detail in future chapters.

Transcription

A gene is complex: it contains not only the code for the resulting protein but also several regulatory factors that determine if and when the region that codes for a protein are read to create protein. What follows is a diagram of the components of a gene that are used in transcription.

For this chapter, we focus on the DNA and the ending product of transcription: mRNA.

Given a specific DNA strand, what is the sequence of the resulting mRNA molecule? We will learn about how mRNA is created in a later chapter.

Translation

Translation involves different types of RNA, and we will explain them in more detail in a later chapter: rRNA, tRNA, mRNA, and microRNA.

After an mRNA is created, it leaves the nucleus and is attracted to or attracts a ribosome, which is a molecule made of rRNA and polypeptides. Then, in the ribosome, and with the assistance of tRNAs, the mRNA is read and an amino acid sequence is created.

DNA and mRNA create sequences with just four types of bases; yet, these bases code for 20 unique amino acids (the makeup of protein). How is this possible? Watch the following video to find out!

The mRNA is read in sets of three bases known as codons. Each codon codes for a single amino acid. In this way, the mRNA is read and the protein product is made.

Below is a chart showing which codons code for which bases. There are two representations; move to the next slide for the second representation.

These charts can be a little confusing at first. Watch the following video to learn how to interpret both chart formats.

This chapter focused on DNA, mRNA, and protein sequences. The next several chapters describe the processes that take place during protein synthesis. Master how sequences are read during protein synthesis (the focus of the current chapter) before moving on to the next chapter. Below are some sources to help further your understanding!

Check out Learn.Genetics’ “How a Firefly’s Tail Makes Light” video for an overview of protein synthesis!

Need a little more practice?

Try out Learn.Genetics’ “Transcribe and Translate a Gene” and The Concord Consortium’s “ DNA to Protein ” interactives for further practice!

Attributions

This chapter is a modified derivative of the following articles:

“ Gene ” by National Human Genome Research Institute, National Institutes of Health, Talking Glossary of Genetic Terms. 

“Nucleic Acids” by OpenStax College,  Biology 2e , CC BY 4.0. Download the original article at https://openstax.org/books/biology-2e/pages/3-5-nucleic-acids

Media Attributions

• Central Dogma © Andrea Bierema
• DNA and RNA © Sponk is licensed under a CC BY-SA (Attribution ShareAlike) license

An Interactive Introduction to Organismal and Molecular Biology Copyright © 2021 by Andrea Bierema is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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11.4 Protein Synthesis (Translation)

Learning objectives.

By the end of this section, you will be able to:

• Describe the genetic code and explain why it is considered almost universal
• Explain the process of translation and the functions of the molecular machinery of translation
• Compare translation in eukaryotes and prokaryotes

The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation , or protein synthesis , the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.

The Genetic Code

Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring amino acids . Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon . The relationship between an mRNA codon and its corresponding amino acid is called the genetic code .

The three-nucleotide code means that there is a total of 64 possible combinations (4 3 , with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon ( Figure 11.12 ). This redundancy in the genetic code is called degeneracy . Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobble position , is less critical. In some cases, if the nucleotide in the third position is changed, the same amino acid is still incorporated.

Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codon s or nonsense codon s . Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the start codon to initiate translation. The reading frame , the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.

The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon.

Check Your Understanding

• How many bases are in each codon?
• What amino acid is coded for by the codon AAU?
• What happens when a stop codon is reached?

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation . The composition of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozyme s) and structural rRNA s, as well as many distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts . Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation . In E. coli , the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).

Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a polyribosome (or polysome ). In both bacteria and archaea , before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes ( Figure 11.13 ). The reason why transcription and translation can occur simultaneously is because both of these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.

Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.

Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other ( Figure 11.14 ). This shape positions the amino-acid binding site, called the CCA amino acid binding end , which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.

An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetase s. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA , and AMP is released.

• Describe the structure and composition of the prokaryotic ribosome.
• In what direction is the mRNA template read?
• Describe the structure and function of a tRNA.

The Mechanism of Protein Synthesis

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli , a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli , this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N -formyl-methionine (fMet-tRNA fMet ) ( Figure 11.15 ). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli . In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

• The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
• Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli , the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNA fMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase , an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 11.16 and listed in Figure 11.17 .

Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational modifications include:

• removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular compartment
• proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins, into a distinct three-dimensional structure
• proteolytic processing of an inactive polypeptide to release an active protein component, and
• various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.
• What are the components of the initiation complex for translation in prokaryotes?
• What are two differences between initiation of prokaryotic and eukaryotic translation?
• What occurs at each of the three active sites of the ribosome?
• What causes termination of translation?

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• Authors: Nina Parker, Mark Schneegurt, Anh-Hue Thi Tu, Philip Lister, Brian M. Forster
• Publisher/website: OpenStax
• Book title: Microbiology
• Publication date: Nov 1, 2016
• Location: Houston, Texas
• Book URL: https://openstax.org/books/microbiology/pages/1-introduction
• Section URL: https://openstax.org/books/microbiology/pages/11-4-protein-synthesis-translation

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What Is Transcription: Understanding the Biological Process

Transcription is the process of converting DNA to RNA in the cell nucleus. This biological process involves RNA polymerase reading the DNA template strand in a 3' to 5' direction , synthesizing complementary RNA in a 5' to 3' direction . Transcription is essential for gene expression and protein production. Understanding what transcription is helps explain how genetic information is transferred from DNA to RNA within cells.

Key aspects of transcription

DNA to RNA conversion : Transcription in biology converts the genetic information in DNA to RNA, specifically messenger RNA (mRNA)

Location : What is transcription? It's a process that occurs in the nucleus of eukaryotic cells

Enzyme involved : RNA polymerase is the primary enzyme responsible for transcription, a key component of the transcription definition

Direction : In transcription, RNA is synthesized in a 5' to 3' direction , while the DNA template is read in a 3' to 5' direction

Types of RNA produced : What is transcription's output? It produces various types of RNA, including mRNA , rRNA , and tRNA

Transcription process

Promoter recognition : In transcription, RNA polymerase, along with transcription factors, recognizes and binds to the promoter region of the gene

Complex formation : The transcription definition in eukaryotes includes a pre-initiation complex (PIC) forming at the promoter, involving various factors like TBP and TAFs

DNA melting : During transcription in biology, the DNA double helix is unwound and separated into single strands at the transcription start site

Nucleotide addition : What is transcription's main action? RNA polymerase adds complementary ribonucleotides to the growing RNA chain

Rate of synthesis : Transcription progresses at a rate of about 50 nucleotides per second in prokaryotes

Proofreading : In transcription, RNA polymerase has some ability to correct errors, though less efficient than DNA polymerase

Termination

Signal recognition : What is transcription's end signal? RNA polymerase recognizes specific termination signals in the DNA sequence

RNA release : In the final step of transcription, the newly synthesized RNA molecule is released from the template DNA strand

Regulation of transcription

Transcription factors : The transcription definition includes proteins that bind to specific DNA sequences to control gene expression

Chromatin structure : In transcription biology, the organization of DNA and proteins affects gene accessibility

Post-transcriptional modifications : What is transcription followed by? RNA can undergo various modifications after transcription, affecting its stability and function

Differences between prokaryotic and eukaryotic transcription

Enzyme complexity : What is transcription like in eukaryotes? They have three different RNA polymerases (I, II, III), while prokaryotes have only one

Transcription factors : The transcription definition for eukaryotes includes more complex transcription factor assemblies compared to prokaryotes

Coupling with translation : In prokaryotic transcription, transcription and translation can occur simultaneously, while in eukaryotes, they are separated by the nuclear membrane

What is transcription in biology?

Transcription in biology is the process of converting DNA to RNA in the cell nucleus. It involves RNA polymerase reading the DNA template strand and synthesizing complementary RNA. This process is essential for gene expression and protein production.

What is the basic transcription definition?

The basic transcription definition is the conversion of genetic information from DNA to RNA. Specifically, it's the synthesis of RNA using DNA as a template, carried out by the enzyme RNA polymerase in the nucleus of eukaryotic cells.

What are the key steps in transcription?

The key steps in transcription are initiation, elongation, and termination. Initiation involves RNA polymerase binding to the promoter region. Elongation is the process of adding complementary ribonucleotides to the growing RNA chain. Termination occurs when the RNA polymerase recognizes specific signals and releases the newly synthesized RNA.

How does transcription differ between prokaryotes and eukaryotes?

In prokaryotes, transcription uses a single RNA polymerase and can occur simultaneously with translation. Eukaryotes have three different RNA polymerases, more complex transcription factor assemblies, and transcription is separated from translation by the nuclear membrane.

What is the role of transcription factors in transcription?

Transcription factors are proteins that bind to specific DNA sequences to control gene expression. They play a crucial role in regulating transcription by either promoting or inhibiting the recruitment of RNA polymerase to specific genes, thus influencing which genes are transcribed and when.

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Article Contents

Lay summary, introduction, materials and methods, data availability, supplementary data, acknowledgements, enl reads histone β-hydroxybutyrylation to modulate gene transcription.

The first four authors should be regarded as Joint First Authors.

• Article contents
• Figures & tables
• Supplementary Data

Chen Chen, Cong Chen, Aiyuan Wang, Zixin Jiang, Fei Zhao, Yanan Li, Yue Han, Ziping Niu, Shanshan Tian, Xue Bai, Kai Zhang, Guijin Zhai, ENL reads histone β-hydroxybutyrylation to modulate gene transcription, Nucleic Acids Research , Volume 52, Issue 17, 23 September 2024, Pages 10029–10039, https://doi.org/10.1093/nar/gkae504

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Histone modifications are typically recognized by chromatin-binding protein modules (referred to as ‘readers’) to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drive cell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone β-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

Elucidating the binding partners of histone post-translational modifications (hPTMs) is key to understanding epigenetic regulatory pathways. Lysine β-hydroxybutyrylation (Kbhb) is a novel hPTM that couples metabolism to transcription. However, the effectors reading this mark are poorly understood as the Kbhb-mediated protein–protein interactions are weak and transient. Here, we presented a quantitative chemical proteomics approach using multivalent photoaffinity probes to robustly capture interactors of this mark. Thus, we identified ENL as a novel binder of Kbhb of histone H3 lysine 9 (H3K9bhb). Biochemical studies and CUT&Tag analysis further revealed that ENL recognizes H3K9bhb and co-localizes with it on gene promoters to modulate transcription and tumorigenesis. This study highlights ENL as a histone Kbhb reader for the regulation of transcription.

Histone post-translational modifications (hPTMs, e.g. lysine acetylation and methylation) are a group of fundamental epigenetic marks found in mammalian chromatin ( 1 , 2 ). With the development of mass spectrometry-based proteomics, a range of novel hPTMs has been reported, including various types of lysine acylation, such as crotonylation (Kcr) ( 3 ), succinylation (Ksucc) ( 4 ), 2-hydroxyisobutyrylation (Khib) ( 5 ), β-hydroxybutyrylation (Kbhb) ( 6 ) and lactylation (Kla) ( 7 ). Emerging evidence suggests that these hPTMs possess important roles in regulating cell processes, including gene transcription, DNA damage repair, replication and chromatin remodeling ( 8–11 ). Moreover, the hPTMs are typically considered as docking sites to execute these functions by recruiting various specific binding partners, referred to as readers ( 12–14 ). Following the first discovery of the bromodomain (BrD) as a reading module of acetyllysine ( 15 ), a number of readers such as YEATS, DPF and ZZ domains have been characterized for type- and site-specific readout of histone acetylation ( 16–20 ). However, downstream effectors capable of interpreting histone non-acetyl acylations such as β-hydroxybutyrylation are poorly understood.

Histone β-hydroxybutyrylation is a novel epigenetic regulatory mark that links metabolism to gene transcription. It is remarkably induced by β-hydroxybutyrate that is derived from starvation or diabetic ketosis of cells where cellular metabolism typically switches and the new steady-state balance exists ( 21 ). The mark may lead to particular transcription events that govern cell growth and development through associating with a set of up-regulated genes that distinguish histone acetylation, corroborating that histone Kbhb has distinct roles from histone Kac ( 6 ). Indeed, Zhang and co-workers ( 22 ) recently reported that BDH1-mediated histone Kbhb up-regulated stemness-associated genes, promoting propagation of hepatocellular carcinoma (HCC) cells. Similarly, the Huang group ( 23 ) uncovered that ketogenesis-generated histone Kbhb enhances gene expression and CD8 + T-cell memory development, coupling epigenetic modification with energy metabolism. In addition, Zheng and colleagues ( 24 ) showed that histone Kbhb regulates ferroptosis-suppressor gene expression in pancreatic acinar cells. However, how histone Kbhb regulates transcription of these gene remains largely elusive. Given the unique structure of Kbhb, traditional histone readers may lack sufficient ability to recognize this hPTM. It is one of the main reasons why readers of Kbhb have been unclear up to now. Thus, identification of readers that specifically recognize histone Kbhb is vital to understand the molecular mechanism of this epigenetic regulation.

Identification of hPTM effector proteins is challenging owing to the fact that the hPTM-mediated protein–protein interactions (PPIs) are weak and transient ( 25 , 26 ). Therefore, an affinity purification approach based on a mimic peptide ( 27 ) may not be applicable for the mining of hPTM readers, whereas photoaffinity probes can be an alternative method ( 28 , 29 ). We recently developed a self-assembled multivalent photoaffinity probe that we used for efficient and selective enrichment of histone Kcr readers and Khib erasers ( 30 , 31 ). In this study, we further design the probe and combine it with a quantitative proteomics approach to selectively profile binders of histone Kbhb. By using this method, the interactomes of β-hydroxybutyrylation of histone H3 lysine 9 (H3K9bhb) were determined, and ENL was identified as a novel H3K9bhb reader. Biochemical and molecular recognition studies and CUT&Tag analysis demonstrate that ENL recognizes H3K9bhb, and co-localizes with this mark on gene promoters. Disrupting the association between ENL and histone Kbhb by structure-guided mutation of Y78A in the YEATS domain reduced the recruitments of ENL to H3K9bhb-enriched peaks, resulting in the down-regulation of genes such as MYC that are responsible for tumorigenesis.

Materials and instruments

Histone peptides with and without lysine trimetylation or β-hydroxybutyrylation modification were synthesized and purified by high-performance liquid chromatography (HPLC) by Beijing SciLight Biotechnology Co Ltd. Thiolated polyethylene glycol (HS-PEG) modified with benzophenone (HS-PEG-Bpa) was obtained from ToYong Biotech Ltd. Gold nanoparticles were purchased from BBI solutions. HPLC solvents used for mass spectrometric analysis of proteins were purchased from Thermo Fisher Scientific Ltd. Antibodies against ENL were obtained from Abcam. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) spectra were acquired by an Autoflex III TOF/TOF mass spectrometer (Bruker), and protein samples were identified by a Nano-LC-Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Synthesis and characterization of hPTM probes

For the fabrication of the probes, 2 mM peptides with or without PTMs and 1 mM HS-PEG-Bpa were mixed and added to the solution of gold nanoparticles (200 μl), and the mixture was shaken at room temperature for 12 h. Then, the functionalized gold nanoparticles were washed with H 2 O (50 μl) twice and the fabricated probes were stored at 4°C for further use. MALDI-TOF-MS and transmission electron microscopy (TEM) were used to confirm the probes.

Extraction of nuclear proteins

Nuclear proteins were extracted from cells using nuclear extraction reagents (Nucleoprotein Extraction Kit, Sangon Biotech). Briefly, HepG2 cells were washed twice with phosphate-buffered saline (PBS; 0.1 M phosphate, 0.15 M NaCl, pH 7.2) and harvested into a tube (1.5 ml). After removing PBS, hypotonic buffer (protease inhibitor added beforehand) was added and incubated for 10 min on ice. Then, the solution was centrifuged at 800 g for 5 min and the supernatant was discarded. The precipitate was washed with hypotonic buffer in a mixer for 30 s followed by centrifugation at 2500 g for another 5 min. After centrifugation, the supernatant was removed. Next, the precipitate was resuspended in lysis buffer and incubated for 20 min. Finally, nucleoprotein was collected by centrifugation at 20 000 g for 10 min.

Enrichment of endogenous binders by hPTM probes

The functionalized probes H3K9bhb and H3K9 were each incubated with 0.5 mg of nuclear extracts for 12 h at 4°C in a rotation wheel. Then, the mixture was irradiated at 365 nm on ice for 20 min using UVP Crosslinker (Analytik Jena). After centrifugation, the precipitate was washed twice with wash buffer 1 (4 M urea in PBS), followed by washing once with buffer 2 (50 mM Tris–HCl, pH 7.8, 200 mM NaCl, 2.5 mM KCl, 2.5 mM MgCl 2 , 1 mM ZnCl 2 ) and once with wash buffer 3 (100 mM NH 4 HCO 3 ). Next, the pellet was suspended in 50 μl of NH 4 HCO 3 (100 mM), and trypsin (1 μg) was added to digest it overnight at 37°C. Finally, the mixture was centrifuged for 2 min and the supernatant was collected. Then, 5 mM dithiothreitol (DTT) was added and incubated with the obtained solution for 1 h at 65°C, followed by reaction with 15 mM iodoacetamide for 45 min and 30 mM cysteine for 30 min at room temperature. The resulting peptides were desalted with C18 material and further analyzed by HPLC-MS/MS.

HPLC-MS/MS analysis

Each tryptic digest was redissolved in 7 μl of HPLC buffer A [0.1% (v/v) formic acid in water]. After centrifugation at 12 000 g for 2 min, the supernatant (5 μl) was injected into a Nano-LC system (EASY-nLC 1200, Thermo Fisher Scientific). Each sample was separated by a C18 column (50 μm inner diameter × 15 cm, 2 μm C18) with a 60 min HPLC gradient at a flow rate of 300 nl/min. The HPLC eluate was electrosprayed directly into an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). The source was operated at 2.2 kV. The mass spectrometric analysis was carried out in a data-dependent mode with an automatic switch between a full MS scan and an MS/MS scan in the orbitrap. For full MS survey scan, the automatic gain control (AGC) target was 1e6, and the scan range was from 350 to 1750 with a resolution of 70 000. The 10 most intense peaks with charge state 2 and above were selected for fragmentation by higher energy collision dissociation (HCD) with a normalized collision energy of 27%. The MS2 spectra were acquired with 17 500 resolution. The exclusion duration for the data-dependent scan was 10 s, and the exclusion window was set at 1.6 Da.

Data processing

The resulting MS/MS data were searched using Proteome Discoverer software (v2.1) with an overall false discovery rate (FDR) for peptides of <1%. Proteins demonstrating an average score ( n  = 3) of <4 were removed from the identification list. Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Carbamidomethylation on cysteine was specified as a fixed modification. Oxidation of methionine and acetylation on the protein N-terminus were set as variable modifications. Mass tolerances for precursor ions were set at ±10 ppm for precursor ions and ±0.02 Da for MS/MS.

Western blot analysis

The enriched proteins were loaded and separated on an 8% sodium dodecylsulfate–polyacrylamide electrophoresis (SDS–PAGE) gel and transferred to a nitrocellulose membrane (Pall Corporation, 0.22 μm). The membranes were first blocked with 5% non-fat milk and incubated with primary antibodies (ENL) overnight at 4°C at a dilution of 1:500. After washing, the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 2 h at room temperature.

AutoDock analysis

AutoDock 4.0 was used to dock the peptides H3K9bhb and H3K9ac into the structures of ENL. The structure of ENL was taken from the Protein Data Bank (PDB) (PDB: 5J9S). Before the docking simulation, the peptide was placed into the middle of the ENL surface as the start point of docking. The parameters for docking were set as follows: the Lamarckian genetic algorithm (LGA) runs were set at 100, and the maximum number of energy evaluations was set at 25 million. The simulation box was fixed at the center of the substrate and the box size was set at 80 Å in all three dimensions. The conformation with the highest binding energy of small molecules was considered as the best conformation.

Recombinant protein expression and purification

Recombinant ENL YEATS (residues 1–148) was cloned into the ET28b-SUMO vector and expressed with an N-terminal 6× His-SUMO tag in Escherichia coli strain BL21(DE3) (ZOMANBIO) and induced overnight by 0.5 mM isopropyl-β- d -thiogalactoside at 16°C in LB medium. Overnight-induced cells were collected and suspended in lysis buffer: 50 mM Tris–HCl, pH 8.0, 500 mM NaCl and 1 mM phenylmethylsulfonyl fluoride (PMSF). Then the cells were lysed using an ultrasonic crusher. After centrifugation, the supernatant was applied to a nickel column, and proteins were eluted with 300 mM imidazole. The resultant proteins were treated with ULP enzyme overnight for His-SUMO tag removal. The tag-free ENL YEATS proteins were further concentrated to ∼8 mg/ml, and stored at −80°C. ENL YEATS mutants were generated using QuikChange (Genstar) methods and verified by sequencing. Recombinant mutant ENL YEATS proteins were expressed and purified with essentially the same method as for wild-type (WT) ENL YEATS .

Isothermal titration calorimetry (ITC)

The ITC experiments were carried out on the MicroCal PEAQ ITC instrument (Malvern Instrument) at 15°C. The protein at 100 μM was dropped into the 1000 μM peptide segment for 19 consecutive drops, and the resulting titration curve was drawn using the ‘one set of binding sites’ model and the Origin 7.0 program. The protein concentration was determined by UV absorption at 280 nm. The peptide concentration was measured by nanodrop.

Analytical gel filtration

Analytical gel filtration experiments for the detection of protein–peptide interactions were carried out using an analytical Superdex S200 (Cytiva) gel filtration column with a flow rate of 0.5 ml/min on an Äkta purifier system (GE Healthcare) in a 20 mM HEPES pH 7.5, 150 mM NaCl buffer at 4°C. The protein and peptide H3K9bhb were incubated at a ratio of 1:1.5 at 4°C and the total applied sample volumes were 300 μl in both cases. Protein elution was followed by recording the UV adsorption at 280 nm.

Fluorescence microscopy

After transfecting with WT-ENL or its mutants, and growth for 48 h, cells were washed in PBS in triplicate followed by being fixed for 15 min in PBS containing 4% (w/v) paraformaldehyde. After washing, the HepG2 cells were then permeabilized for 30 min with PBS containing 0.25% Triton X-100, blocked with 3% bovine serum albumin (BSA) in PBS for 2 h in room temperature, and incubated with mouse anti-DDDDK-Tag monoclonal antibody (Abclonal, AE005) and H3K9bhb (PTMBio, PTM-1250RM) antibody overnight at 4°C. After washing with PBS, cells were incubated with secondary antibodies coupled to AlexaFluor 488 or 647 for 1.5 h in room temperature, and then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, cells were imaged using a Zeiss LSM 800 microscope with a ×63 oil objective. Images were merged using Adobe Photoshop and analyzed by ImageJ.

CUT&Tag

CUT&Tag is operated according to the manufacturer's instructions for the Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme Biotech). In brief, HepG2 cells expressing WT-ENL or Mutant-ENL were collected and counted for incubation with pre-treated concanavalin A (ConA) beads. Subsequently, cells were resuspended in an antibody buffer and incubated overnight at 4°C with the corresponding primary antibody of Flag or anti-H3K9bhb. The secondary antibodies were diluted in appropriate proportions and incubated with the cells at room temperature. Subsequently, pA/G-Tnp transposons were rotated and incubated with the samples for 1 h to activate the translocase fragment DNA and extract the DNA. The DNA library was constructed with the TruePreP Index Kit V2 for Illumina (TD202, Vazyme Biotech). The library was purified by VAHTS DNA Clean Beads (N411, Vazyme Biotech) and sequenced by Illumina novaseq 150PE.

ChIP-qPCR analysis

Chromatin precipitation (ChIP) analysis was performed essentially as described below. Briefly, HepG2 cells were cross-linked with 1% formaldehyde for 10 min and stopped with 125 mM glycine. The isolated nuclei were resuspended in nuclei lysis buffer and sonicated using a Bioruptor Sonicator (Diagenode). The samples were immunoprecipitated with 2–4 μg of the appropriate antibodies overnight at 4°C. Protein A/G beads were added and incubated for 1 h, and the immunoprecipitates were washed twice each with low-salt, high-salt and LiCl buffers. Eluted DNA was reverse-cross-linked, purified using a polymerase chain reaction (PCR) purification kit (Genestar) and analyzed by quantitative real-time PCR on the ABI 7500-FAST System using the Power SYBR Green PCR Master Mix (Genestar). Statistical differences were calculated using a two-way unpaired Student's t -test. The primers used for qPCR are listed in Supplementary Table S3 .

RNA-seq analysis

RNA-seq samples were sequenced using the Illumina Hiseq 2500, and raw reads were mapped to the human reference genome (hg19) and transcriptome using the RNA-seq unified mapper. Read counts for each transcript were calculated using HTseq v0.6.1 employing default parameters 54. Differential gene expression analyses were performed using the ‘exactTest’ function in edgeR v3.055. Gene Ontology (GO) analysis was performed using the DAVID Bioinformatics Resource 6.756. The volcano plot was drawn by using the ggplot2 package ( https://cran.r-project.org/package=ggplot2 ) in the R computing environment.

Cell Counting Kit-8 (CCK-8) assay

Cell Counting Kit-8 (CCK-8) was used to measure cell proliferation. HepG2 cells were seeded into 96-well plates at a density of 3000 cells per well. Every 24 h for 7 days, 10 μl of the CCK-8 solution was added to each well with incubation for 2 h at 37°C. The absorbance of each well was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific). Finally, the numbers of living cells over 5 days were plotted in a graph to calculate the cell proliferation rate.

Cell migration assay

The migration assays were performed using a transwell chamber. After transfection for 24 h, HepG2 cells were seeded in transwell chambers at 7 × 10 4 cells per well. Forty hours later, the cells which had migrated through the filter were fixed and stained with 2% crystal violet. For each chamber, the mean of the migrated cell number from five randomly fields was calculated and displayed in the plot.

Development of a quantitative chemical proteomics approach for mapping hPTM binders

In order to selectively and robustly identify hPTM interactors, we envisaged a chemoproteomics strategy that coupled a multivalent photoaffinity probe with quantitative proteomics. We designed probe H3K9bhb that contains Kbhb-installed peptide (mimic histone Kbhb) and photo-cross-linker-linked PEG, based on the spacer of the cross-linker and the mark, leading to improvement in the efficiency of target identification. Furthermore, the multivalent effect of the photo-cross-linker produces multiple labeling events contributing to capture of weak PPIs. Based on the above, we prepared probe H3K9bhb through a self-assembled technique while a control probe H3K9, containing lysine 9 instead of Kbhb, was also synthesized to minimize non-specific binders and improve the determination of H3K9bhb interatomes (Figure 1A , B ).

Proteomic profiling of hPTM interactors using photoaffinity probes. ( A ) Schematic workflow for enrichment and identification of hPTM target proteins by comparative proteomics along with self-assembly multivalent photoaffinity probes. ( B ) Structure of lysine β-hydroxybutyrylation. AuNPs, gold nanoparticles; PEG, polyethylene glycol. ( C ) Volcano plot of protein enrichment ratio and statistical analysis from proteomic experiments ( n  = 3) by using probes H3K4me3 and H3K4. Blue hits indicate that proteins meet the criteria (ratio ≥ 2.0 and P -value ≤ 0.05). ( D ) Several known H3K4me3 reader proteins identified with high confidence.

To test whether the strategy could be used to identify hPTM readers, we first set up comparative proteomic experiments to capture target proteins of H3K4me3, a well-established histone mark associated with actively transcribed genes ( 32 ). Similar to H3K9bhb, probe H3K4me3 and probe H3K4 were, respectively, prepared and incubated with cell lysate, and subsequently subjected to UV irradiation by which cross-linking between the probe and its targets could occur. After removing non-specific binders by stringent washing, the captured proteins were enriched by centrifugation and digested by on-bead trypsinization, followed by detection with HPLC-MS/MS to quantify protein abundance. Finally, the spectral counts ( 33 , 34 ) detected for each protein were used to calculate the enrichment ratios for proteins identified by probes H3K4me3 and H3K4 (Figure 1A ). We performed three biological replicates to reliably identify binders enriched by probe H3K4me3, and proteins with a maximal mean of < 2.0 spectral counts were discarded. For the remaining proteins, we used an averaged enrichment ratio (H3K4me3/H3K4) of 2.0 ( n  = 3) as the cut-off to exclude non-specific binding proteins. Furthermore, a t -test between probe H3K4me3 and H3K4 was conducted to generate a set of proteins meeting a P -value < 0.05 as high-confidence targets. The identified proteins were then displayed by volcano plots as (log 2 ) of the enrichment ratio against their statistical significance [–log 10 ( P -value)] (Figure 1C ; Supplementary Table S1 ).

As a result, 43 protein hits that meet the above criteria were identified, and the GO analysis on these proteins demonstrated that prominent among them are those enriched in chromatin binding, transcription factor binding and transcription regulator activity ( Supplementary Figure S1 ), demonstrating that these proteins may be read by H3K4me3 to mediate cell functions such as gene expression. As expected, our analysis of proteins identified by the probe found several known readers of H3K4me3, such as TAF3 ( 35 ), PHF8 ( 36 ) and CHD1 ( 37 ), displaying 2.5- to 64.3-fold preference for the probe H3K4me3 over probe H3K4 (Figure 1D ). The result demonstrates that our approach is capable of identifying endogenous HPTM readers from cell lysates.

Identification of ENL as an H3K9bhb binder through the chemoproteomic approach

We next applied this validated chemoproteomics strategy to identify targets of H3K9bhb, a histone mark associated with gene transcription ( 22 ). Recently, the enzymes that are responsible for removing or installing this modification have been discovered ( 38 , 39 ), while readers which prefer this mark remain largely unknown. Thus, it is of great importance to mine readers of H3K9bhb in order to decipher its biological effects. Herein, we developed probe H3K9bhb and H3K9, confirmed by MS and TEM data ( Supplementary Figures S2 and S3 ), to uncover putative binders of the mark in cellular lysates. The probe H3K9bhb or H3K9 was first incubated with cell extracts, followed by UV-irradiation and stringent washing to remove non-specific binding proteins. Then, the enriched proteins were digested and subjected to LC-MS/MS analysis. Similarly, to exclude the false-positive binders, we also generated a volcano plot and screened proteins with a fold enrichment (probe H3K9bhb/H3K9) ≥2 and a P -value ≤ 0.05. As shown in Figure 2A and Supplementary Table S2 , 43 proteins, identified from the pulldowns by probe H3K9bhb, satisfied the criteria. We next employed GO analysis on these potential binding proteins regarding their molecular function distributions, and found that major proteins are enriched with nucleic acid binding, catalytic activity and transcription regulator activity (Figure 2B ).

Identification of ENL as a H3K9hbb-binding protein using a chemoproteomics approach. ( A ) Volcano plot of protein fold enrichment and P -value from proteomic experiments ( n  = 3) by using probes H3K9hbb and H3K9. Red hits are proteins with a ratio ≥ 2.0 and P -value ≤ 0.05.( B ) Molecular function analysis of H3K9bhb targets (red hits in A) by GO. ( C ) Parallel reaction monitoring (PRM) mass spectrometry and ( D ) western blotting analysis for the captured binders by probes H3K9hbb and H3K9. ( E ) ITC fitting curves of the ENL titrated with unmodified (H3K9un) or β-hydroxybutyryl histone H31-17 peptides (H3K9bhb). ( F and G ) Molecular recognition analysis of H3K9bhb read by ENL. Electrostatic potential surface view of the ENL space filled by H3K9bhb peptide. Bottom right, close-up view of the H3K9bhb-binding pocket of ENL (F). ( G ) The bonding network between H3K9bhb peptide and ENL. Hydrogen bonds are shown as green dashes. Key residues of ENL are depicted as blue sticks and labeled in orange; the H3K9bhb peptide is shown as yellow sticks and labeled in black.

Considering that nucleic acid binding was the top-ranked pathway and the related proteins may function through binding to histones, we continued to focus on the proteins involved in this cluster. After ruling out several RNA-binding proteins, we further concentrated on four targets (ZNF787, TFCP2, ENL and GTF2H4) that are associated with chromatin binding. Further domain analysis indicated that only ENL possesses the binding modules of acetylated histone ( 40 ); we therefore speculated that the H3K9bhb mark may be recognized by ENL, and selected it for further investigation.

ENL efficiently binds to H3K9bhb through its YEATS domain in vitro

We next decided to investigate the putative interaction between ENL and the mark by using parallel reaction monitoring (PRM) MS, which has been reported as a targeted proteomic method for accurate quantitative measurement of proteins ( 41 ). Our result revealed that ENL exhibits a 3.9-fold enrichment for probe H3K9bhb compared with probe H3K9 (Figure 2C ), which is in line with the proteomics profiling data. In addition, analytical gel filtration of ENL with or without peptide H3K9bhb showed two obvious separating peaks ( Supplementary Figure S4 ), suggesting the binding of ENL and H3K9bhb. Next, to further confirm ENL–H3K9bhb interaction, we performed photoaffinity enrichment from a cell lysate followed by western blotting analysis of proteins isolated by probes H3K9bhb and H3K9, respectively. As expected, probe H3K9bhb captured ENL more efficiently than probe H3K9, validating that ENL could directly and selectively interact with H3K9bhb (Figure 2D ).

In light of the ENL YEATS domain being a reader of histone acetylation ( 40 ), we proposed that ENL might recognize H3K9bhb via its YEATS domain. To test this hypothesis, we recombinantly expressed the YEATS domain of ENL (ENL YEATS ), and performed an ITC assay using histone H3K14 (1–17) peptides, with binding dissociation constants ( K d ) of 0.71 μM, 8.44 μM and not detected (ND) for H3K9bhb, H3K9ac and unmodified peptides, respectively (Figure 2E ; Supplementary Figure S5A ). Notably, compared with H3K9ac, ENL YEATS revealed an ∼12-fold increase in affinity for H3K9bhb. Furthermore, pulldown assays using biotinylated peptides showed that the YEATS domain of ENL exhibited a higher binding affinity toward H3K9bhb than H3K9ac ( Supplementary Figure S5B ). In addition, we also implemented ITC detection using H3K9bhb peptide with other YEATS domain-containing proteins (AF9, GAS41 and YEATS2), and measured a K d of 11.0 μM, 6.7 μM and ND, respectively ( Supplementary Figure S5C ), which indicated that H3K9bhb may selectively recognize YEATS module proteins. Together, these results established that ENL favorably bound to H3K9bhb via its YEATS domain.

Moreover, to decipher the molecular basis for how ENL recognizes the H3K9bhb mark, we docked the binding of ENL YEATS (PDB: 5J9S) with H3K9bhb peptide. As shown in Figure 2F and  G , H3K9bhb inserts into the binding pocket formed with ENL YEATS residues, such as H56, S58, F59 and Y78, in which an aromatic sandwich cage for K9bhb recognition is clamped by aromatic residues such as F59 and Y78. In addition to electrostatic and hydrophobic contacts, the recognition of H3K9bhb peptide by ENL is stabilized through a hydrogen bond of the Y78 residue and the hydroxyl group within β-hydroxybutyrylamide, differing from H3K9ac that is facilitated by a hydrogen bond between acetylamide and the Y78 main chain. This suggested that there may be different mechanisms for recognizing H3K9bhb and H3K9ac, thereby contributing to distinct affinities for ENL ( Supplementary Figures S5D and S6 ). Collectively, these findings demonstrate that ENL is able to preferably recognize H3K9bhb relying on some key residues such as Y78, as a potential reader of this histone mark.

ENL is intracellularly associated with H3K9bhb

To assess the association of ENL with H3K9bhb in cells, we set up ENL mutagenesis and immunofluorescence (IF) assay. Structure-guided mutagenesis was designed to alanine mutation of the Khbb-surrounding residues including S58, F59 and Y78. Subsequently, ITC assays revealed that the affinity of H3K9bhb binding for all mutations (S58A, F59A and Y78A) dramatically dropped in comparison with WT-ENL (Figure 3A ). We then performed IF imaging determination to investigate their co-localization in cells transfected with WT-ENL or FLAG-tagged mutants, followed by reaction with antibodies against FLAG or H3K9bhb. As shown in Figure 3B and  C , FLAG-tagged ENL and H3K9bhb rendered an even distribution pattern in the nucleus, and the signals resulting from them were merged with a high Pearson correlation coefficient of 0.76, thereby validating their co-localization in cells. By contrast, the value was significantly reduced in the ENL mutants, which indicates the disruption of the association owing to the mutation. Together, these observations suggest that ENL interacts with H3K9bhb via key residues of its YEATS domain in cells.

ENL mutagenesis and co-localization studies. ( A ) ITC titration fitting curves of ENL mutants with H3K9bhb peptide. ( B ) Representative IF images of cells treated with antibodies against FLAG (red) or H3K9bhb (green). FLAG-tagged ENL or its mutants were transfected into cells for IF analysis. ( C ) Quantification of the FLAG-ENL or its mutants co-localized with H3K9bhb using Pearson's correlation analysis.

ENL co-localizes with H3K9bhb across the whole genome

ENL is associated with acute myeloid leukemia, but its relationship with HCC remains unclear. Utilizing gene expression profiling interactive analysis (GEPIA), we found that ENL in HCC was remarkably up-regulated, and the high expression level of ENL was linked to short overall survival ( Supplementary Figures S7 and S8 ). Based on this, we proposed that ENL may recognize H3K9bhb to modulate gene transcription, potentiating progression of HCC cells.

To investigate this, we firstly implemented CUT&Tag assays in HepG2 cells to test whether ENL links H3K9bhb in the native chromatin context. The cells stably expressing Flag-tagged ENL were immunoprecipitated using Flag antibodies or H3K9bhb antibodies, respectively. By high-throughput sequencing of these CUT&Tag experiments, we identified 9660 H3K9bhb-enriched peaks and 12 077 ENL-bound peaks, 7232 of which were co-occupied by both H3K9bhb and ENL (Figure 4A ). Notably, the overlapping peaks were mainly positioned within gene promoter regions (62.1%) and others were mainly localized in the intergenic regions such as enhancers (Figure 4B ), suggesting that H3K9bhb may associate with ENL to modulate gene transcription. Furthermore, the heatmap and average distribution of H3K9bhb and ENL in the promoter regions revealed a strong enrichment at transcription start sites (TSSs) and adjacent regions within 3 kb (Figure 4C ). Genome browser views of the CUT&Tag signals of selected genes, which related to tumorigenesis, further confirmed the co-localization of ENL with H3K9bhb in TSSs (Figure 4F ). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that ENL-bound and H3K9bhb-TSSs-marked genes were involved in the regulation of gene transcription, mitogen-acivated protein kinase (MAPK) signaling, focal adhesion, and so on (Figure 4D ).

ENL co-localizes with H3K9bhb across the whole genome through CUT&Tag analysis. ( A ) Venn diagram showing the overlap of H3K9bhb- and ENL-occupied genes in cells. ( B ) The distribution of overlapping peaks within genes. ( C ) The heatmap illustrating that H3K9bhb globally co-localized with ENL at promoter regions. ( D ) The bubble graph showing the enrichment pathways in the KEGG analysis of the overlapping genes in ENL and H3K9bhb CUT&Tag results. ( E ) Averaged genome-wide occupancies of WT-ENL and Mutant-ENL (Y78A) around TSSs. ( F ) The genome browser view of WT-ENL, Mutant-ENL (Y78A) and H3K9bhb at the CCND1, MYC and TRIM71 locus, respectively. The y -axis represents CUT&Tag intensity. ( G ) The quantitative results by ChIP-qPCR assay for the selected genes. Data are shown as means (±SEM) of two independent experiments.

Given that ENL enables recognition of histone acetylation and crotonylation marks, we further explored whether there are genes co-occupied by H3K9ac-ENL, H3K9cr-ENL and H3K9bhb-ENL interactions through CUT&Tag detection. Consequently, we identified 1946 peaks marked by these modifications, among which the peaks were mainly localized at promoter regions and the genes resulting from these peaks were enriched in a variety of functional processes ( Supplementary Figure S9 ). Importantly, there are a number of peaks that are specifically occupied by H3K9bhb-ENL, suggesting potential distinctive outputs mediated by this mark ( Supplementary Figure S9A ). Furthermore, H3K9ac rather than H3K9cr was marked at the H3K9bhb-ENL-enriched genes (such as MYC ) ( Supplementary Figure S9D ), indicating the putative synergistic effect by histone acetylation on H3K9bhb-regulated gene transcription.

To further validate the dependence of ENL-binding peaks on the YEATS domain, we performed CUT&Tag assays with cells expressing Mutant-ENL (Y78A). As shown in Figure 4E , compared with WT-ENL, the average promoter region occupancies of Mutant-ENL were dramatically reduced, suggesting that ENL binds to these gene regions relying on the YEATS domain. Consistently, the Y78A mutation greatly disrupted ENL’s association with H3K9bhb at the selected gene promoter regions shown by ChIP-qPCR analysis (Figure 4F , G ). Together, these findings demonstrate that ENL co-localizes with H3K9bhb across the genome (mainly on gene promoters), while the ENL YEATS mutation attenuates the correlation.

ENL interacts with H3K9bhb to regulate gene transcription depending on the YEATS domain

It was previously reported that ENL could activate oncogenic gene transcription through the link with histone acetylation. Given the association between ENL and H3K9bhb at gene promoters, we wondered if the ENL–H3K9bhb interaction could regulate gene expression. To this end, we performed RNA-seq by ectopically expressing WT-ENL or Mutant-ENL in HepG2 cells. Through the cut-off of log 2 (fold change)>1 and P -value < 0.05, our result showed that the Mutant-ENL leads to suppressed expression of 583 genes (mainly included in the regulation of lysine writer activity, RNA binding, and so on) (Figure 5A , B ), suggesting that ENL could be recruited to these genes to modulate transcription via the YEATS domain.

ENL correlates with H3K9bhb to regulate gene expression and cell survival relying on the YEATS domain. ( A ) Volcano plots of RNA-seq results of cells expressing WT-ENL compared with cells harboring Mutant-ENL. The up-regulated genes are colored in red, and down-regulated genes are shown in green. ( B ) The bubble graph showing the enrichment pathways in the KEGG analysis of the up-regulated genes in ENL RNA-seq compared with the ENL mutation group. ( C ) Venn diagram displaying the overlap of H3K9bhb-occupied and Mutant-ENL-down-regulated genes in cells. ( D ) The browser view of H3K9bhb CUT&Tag and ENL and its mutant RNA-seq track showing that mutation of ENL leads to reduced occupation at H3K9bhb-enriched TSSs, and down-regulates the expression of CCND1, MYC and TRIM71. ( E ) qRT-PCR analysis of the expression of the indicated genes in cells as in (D). ( F ) Cell proliferation assay in cells expressing WT-ENL or its mutants (Y78A). ( G ) Cell migration ability was determined by transwell assay in cells as in (F). ** P < 0.01; *** P < 0.001. ( H ) The proposed mechanism whereby H3K9bhb recruits ENL to chromatin, and associates with it at gene promoters to boost transcription.

To exclude the potential effect of histone acetylation on the regulation of gene expression, we integrated H3K9bhb-occupied genes with those down-regulated by Mutant-ENL to define the overlapping genes as targets of H3K9bhb-ENL recognition. As a result, 113 genes satisfied this criteria, and responded for both H3K9bhb and ENL (Figure 5C ). As expected, H3K9bhb and ENL CUT&Tag with RNA-seq track displayed that Mutant-ENL at H3K9bhb-enriched promoters results in down-regulation of the selected genes (Figure 5D ). Notably, their transcript levels were verified by reverse transcription–quantitative PCR (RT–qPCR) analysis (Figure 5E ). Furthermore, CCK-8 detection and transwell assay showed that mutation of ENL led to the remarkable reduction of cell proliferation and migration capacity (Figure 5F , G ). Collectively, our results reveal a model where ENL reads H3K9bhb relying on its YEATS domain at gene promoters and recruits other factors to chromatin, to foster expression of genes such as MYC that are crucial for cell proliferation and tumorigenesis (Figure 5H ).

In this study, we developed a quantitative chemoproteomics approach based on multivalent photoaffinity probes to capture hPTM-binding proteins. Using this strategy, we identified a number of known H3K4me3 readers, validating the effectiveness and advantage of the method. Moreover, we extended this strategy to mine the putative binders of H3K9bhb, a newly uncovered histone mark, and for the first time identify ENL as a novel target of this mark.

As a novel epigenetic mark, histone β-hydroxybutyrylation plays a key role in gene transcription. However, the involved regulation mechanism remains largely elusive. Given that histone readers serve as the core element of such epigenetic regulation, the identification of ENL provides a new opportunity to reveal the regulatory mechanism of histone Kbhb. Based on H3K9bhb chemoproteomics findings and biochemical and molecular docking studies, we revealed that ENL is capable of binding to H3K9bhb relying on its YEATS domain, in which the Y78 residue as the key site interacts with the β-hydroxybutyrate group to contribute to the recognition. Indeed, further CUT&Tag coupled with RNA-seq analysis revealed that the ENL-Y78A mutation disrupts the association with H3K9bhb on the promoters of genes such as MYC that are essential for tumorigenesis. Hence mutation of Y78A at ENL in clinical cancer samples may be explored as a promising candidate marker.

ENL has already been reported as a histone acetylation and crotonylation reader that modulates gene transcriptional programs. Here we also queried the candidate targets co-occupied by H3K9ac, H3K9cr, H3K9bhb and ENL, and demonstrated that there are numerous peaks exclusively marked by H3K9bhb-ENL in addition to the overlapping peaks, which indicated the potential distinctive gene transcription events regulated by H3K9bhb. Intriguingly, we also found that H3K9ac was enriched in the gene promoter regions of H3K9bhb-regulated genes such as MYC . Condisidering a higher binding affinity of H3K9bhb ( K d  = 0.71 μM) than H3K9ac ( K d  = 8.44 μM) towards ENL as well as lower peaks marked by H3K9bhb compared with H3K9ac, we surmise that histone Kbhb marks are likely to modulate transcription of these genes by competing for the binding of ENL with Kac, implying that H3K9bhb may function synergistically with H3K9ac through the recognition of ENL.

The work describes a mechanism of histone β-hydroxybutyrylation recruitment of ENL to gene promoters to activate transcription in cancer cells. Further investigations could assess whether the mechanism is available in other cells including starvation-induced liver cells. Future studies are also expected to determine the role of β-hydroxybutyrate in modulating histone Kbhb and thereby boosting cell proliferation.

In principle, we present a general chemoproteomic approach for capture of hPTM targets and identify ENL as a new reader of H3K9bhb. We envisage that our work will be useful for elucidating epigenetic regulatory mechanisms underlying histone marks in cells.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE ( 42 ) partner repository with the dataset identifier PXD051689. RNA-sequencing and CUT&Tag data are available through GEO (GSE266268, 266269).

Supplementary Data are available at NAR Online.

We thank members of the K.Z. laboratory for constructive comments and discussion on this work.

Author Contributions : G.Z. and K.Z. designed the research; Chen C., Cong C., G.Z., Z.J., F.Z., Y.L., Z.N., S.T. and X.B. performed the research; Chen C., G.Z., A.W. and Y.H. analyzed the data; and G.Z. and K.Z. wrote the paper.

This work was financially supported by the National Natural Science Foundation of China [22374106, 22074103 and 22274114]; and the Talent Excellence Program from Tianjin Medical University.

Conflict of interest statement . None declared.

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Transcription factor and protein regulatory network of pmacre1 in pinus massoniana response to pine wilt nematode infection.

1. Introduction

2. materials and methods, 2.1. plant materials, 2.2. cloning and analysis of the pmacre1 gene promoter in p. massoniana, 2.3. detection of pmacre1 gene promoter activity, 2.4. isolation of proteins binding to the pmacre1 gene promoter, 2.5. subcellular localization of proteins binding to the pmacre1 gene promoter, 2.6. inoculation of pine wood nematode to p. massoniana seedlings and proteins extraction, 2.7. co-ip for isolation of pmacre1 interacting proteins, 2.8. kegg enrichment analysis for pmacre1-interacting proteins, 2.9. bifc assay for validating interactions among pmccoaomt, pmcas, and pmacre1, 3.1. pmacre1 gene promoter and its cis-elements, 3.2. activity of the pmacre1 gene promoter, 3.3. dna-binding proteins of the pmacre1 gene promoter, 3.4. subcellular localization of the pmmyb8 transcription factor, 3.5. proteins interacting with pmacre1, 3.6. kegg enrichment analysis of pmacre1 interacting proteins in p. massoniana, 3.7. bifc validation of interactions between pmacre1 and pmccoaomt, pmcas, 4. discussion, supplementary materials, author contributions, data availability statement, conflicts of interest.

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Xie, W.; Lai, X.; Wu, Y.; Li, Z.; Zhu, J.; Huang, Y.; Zhang, F. Transcription Factor and Protein Regulatory Network of PmACRE1 in Pinus massoniana Response to Pine Wilt Nematode Infection. Plants 2024 , 13 , 2672. https://doi.org/10.3390/plants13192672

Xie W, Lai X, Wu Y, Li Z, Zhu J, Huang Y, Zhang F. Transcription Factor and Protein Regulatory Network of PmACRE1 in Pinus massoniana Response to Pine Wilt Nematode Infection. Plants . 2024; 13(19):2672. https://doi.org/10.3390/plants13192672

Xie, Wanfeng, Xiaolin Lai, Yuxiao Wu, Zheyu Li, Jingwen Zhu, Yu Huang, and Feiping Zhang. 2024. "Transcription Factor and Protein Regulatory Network of PmACRE1 in Pinus massoniana Response to Pine Wilt Nematode Infection" Plants 13, no. 19: 2672. https://doi.org/10.3390/plants13192672

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