Chapter 17 Transcription, RNA Processing, and Translation
Chapter 17 Transcription, RNA Processing, and Translation BIOL 2311
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Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu 17.1 An Overview of Transcription First step in converting genetic information into proteins is to synthesize an RNA version of the instructions archived in DNA. o Enzymes called RNA polymerases are responsible for synthesizing mRNA. o NTPs have a hydroxyl group (OH) group on the 2’ carbon. This makes the sugar in an NTP a ribose instead of the deoxyribose sugar of DNA. o Once an NTP that matches a base on the DNA template is in place, RNA polymerase cleaves off two phosphates and catalyzes the formation of a phosphodiester linkage between the 3’ end of the growing mRNA chain and the new ribonucleoside monophosphate. As this 5’ 3’ matchingandcatalysis process continues, an RNA that is complementary to the gene is synthesized. This is transcription. Only one of the two DNA strands is used as a template and transcribed, or “read,” by RNA polymerase. o The strand that is read by the enzyme is the template strand. o The other strand is called the nontemplate strand or coding strand. Coding strand is an appropriate name, because, with one exception, its sequence matches the sequence of the RNA that is transcribed from the template strand and codes for a polypeptide (talking about the RNA strand). The coding strand and RNA don’t match exactly, because RNA has uracil (U) rather than the thymine (T) found in the coding strand. Likewise, an adenine (A) in the DNA template strand specifies a U in the complementary strand. Like DNA polymerases, an RNA polymerase performs a templatedirected synthesis in the 5’ 3’ direction. o But unlike DNA polymerases, RNA polymerases do not require a primer to begin transcription. Initiation: How Does Transcription Begin in Bacteria? Initiation phase of transcription. The enzyme (RNA polymerase) cannot initiate transcription on its own. o A detachable protein subunit called sigma must bind to the polymerase before transcription can begin. Bacterial RNA polymerase + sigma = holoenzyme. o A holoenzyme consists of a core enzyme (RNA polymerase, in this case), which contains the active site for catalysis, and other required proteins (such as sigma). When sigma is added to the polymerase and DNA, the holoenzyme forms and bounds only to specific sections of DNA. o These binding sites were named promoters, because they are sections of DNA that promote the start of transcription. o Sigma was responsible for guiding RNA polymerase to specific locations where transcription should begin. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu Bacterial Promoters Promoters were 4050 base pairs long and had a particular section in common: a series of bases identical or similar to TATAAT. This sixbasepair sequence is now known as the 10 box, because it is centered about 10bases from the point where bacterial RNA polymerase starts transcription. DNA that is located in the direction RNA polymerase moves during transcription is said to be downstream from the point of reference; DNA located in the opposite direction is said to be upstream. o Thus, the 10 box is centered about 10 bases upstream from the transcription start site. The place where transcription begins is called the +1 site. Soon after the discovery of the 10 box, researchers recognized that the sequence TTGACA also occurred in promoters and was about 35 bases upstream from the +1 site. This second key sequence is called the 35 box. Events inside the Holoenzyme In bacteria, transcription begins when sigma, as part of the holoenzyme complex, binds to the 35 and 110 boxes. o Sigma, and not RNA polymerase, makes the initial contact with the DNA of the promoter. o Sigma’s binding to a promoter determines where and in which direction RNA polymerase will start synthesizing RNA. Once the holoenzyme is bound to a promoter for a bacterial gene, the DNA helix is opened by RNA polymerase, creating two separated strands of DNA. o The template strand is threaded through a channel that leads to the active site inside RNA polymerase. Ribonucleoside triphosphates (NTPs)—the RNA building blocks—enter a channel in the enzyme and diffuse to the active site. When incoming NTP pairs with a complementary base on the template strand of DNA, RNA polymerization begins. The initiation phase of transcription is complete as RNA polymerase extends the mRNA from the +1 site. Elongation and Termination Once RNA polymerase begins moving along the DNA template synthesizing RNA, the elongation phase of transcription is under way. RNA polymerase is a macromolecular machine with different parts. o In the interior of the enzyme, a group of amino acids forms a rudder to help steer the template and nontemplate strands through the channels inside the enzyme. o Meanwhile, the enzyme’s active site catalyzes the addition of nucleotides to the 3’ end of the growing RNA molecule at the rate of about 50 nucleotides per second. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu o A group of projecting amino acids forms a region called the zipper to help separate the newly synthesized RNA from the DNA template. Termination ends transcription. In bacteria, transcription stops when RNA polymerase transcribes a DNA sequence that functions as a transcriptiontermination signal. The bases that make up the termination signal in bacteria are transcribed into a stretch of RNA with an important property: As soon as it is synthesized, this portion of the RNA folds back on itself and forms a short double helix that is held together by complementary base pairing. o This type of RNA secondary structure is called a hairpin. o The hairpin structure disrupts the interaction between RNA polymerase and the RNA transcript, resulting in the physical separation of the enzyme and its product. Transcription in Eukaryotes Eukaryotes have three polymerases—RNA polymerase I, II, and III – that are often referred to as pol I, pol II, and pol III. o Each polymerase transcribes only certain types of RNA in eukaryotes. o RNA pol II is the only polymerase that transcribes proteincoding genes. Promoters in eukaryotic DNA are more diverse than bacterial promoters. o Most eukaryotic promoters include a sequence called the TATA box, centered about 30 base pairs upstream of the transcription start site. Instead of using a sigma protein, eukaryotic RNA polymerases recognize promoters using a group of proteins called basal transcription factors. o Basal transcription factors assemble at the promoter, and RNA polymerase follows. Termination of eukaryotic proteincoding genes involves a short sequence called the polyadenylation signal or poly(A) signal. o Soon after the signal is transcribed, the RNA is cut by an enzyme downstream of the poly(a) signal as the polymerase continues to transcribe the DNA template. Bacteria end transcription at a distinct site for each gene, but in eukaryotes, transcription ends variable distances from the poly(A) signal. 17.2 RNA Processing in Eukaryotes In bacteria, when transcription terminates, the result is a mature mRNA that’s ready to be translated into a protein. When eukaryotic genes of any type are transcribed, the initial product is termed a primary transcript. This RNA must undergo multistep processing before it is functional. o For proteincoding genes, the primary transcript is called a premRNA. The Startling Discovery of Split Eukaryotic Genes The regions in a eukaryotic gene that code for proteins are occasionally interrupted by stretches of hundreds or even thousands of intervening bases. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu o Although these intervening bases are part of the gene, they do not code for a product. To make a functional RNA, eukaryotic cells must dispose of certain sequences inside the primary transcript and then combine the separated sections into an integrated whole. The regions of eukaryotic genes that are part of the final mRNA are referred to as exons (because they are expressed). The sections of primary transcript not in mRNA are referred to as introns (because they are intervening). Introns are sections of genes that are not represented in the final RNA product. RNA Splicing The transcription of eukaryotic genes by RNA polymerase generates a primary transcript that contains both exons and introns. o As transcription proceeds, the introns are removed from the growing RNA strand by a process known as splicing. o Pieces of primary transcript are removed and the remaining segments are joined together. o Splicing occurs within the nucleus while transcription is still under way and results in an RNA that contains an uninterrupted genetic message. Splicing of primary transcripts is catalyzed by RNAs called small nuclear RNAs(snRNAs) working with a complex of proteins. These proteinplusRNA macromolecular machines are known as small nuclear ribonucleoproteins, or snRNPs. The snRNAs of the snRNPs recognize RNA sequences critical for splicing. Splicing can be brokeinto four steps. 1. The process begins when snRNPs bind to the 5’ exonintron boundary, which is marked by the bases GU, and to a key adenine ribonucleotide (A) near the end of the intron. 2. Once the initial snRNPs are in place, other snRNPs arrive to form a multipart complex called a spliceosomen. 3. The intron forms a loop plus a singlestranded stem with the adenine at its connecting point. 4. The lariat is cut out, and a phosphodiester linkage links the exons on either side, producing a continuous coding sequence—the mRNA. Both the cutting and rejoining reactions that occur during splicing are catalyzed by the snRNA molecules in the spliceosome—meaning that the reactions are catalyzed by a ribozyme (RNA molecule that acts as a catalyst). Adding Caps and Tails to Transcripts As soon as the 5’ end of a eukaryotic premRNA emerges from RNA polymerase, enzymes add a structure called the 5’ cap. The cap consists of a modified guanine nucleotide with three phosphate groups. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu An enzyme cleaves the 3’ end of the premRNA downstream of the poly (A) signal. Another enzyme adds a long row of 100250 adenine nucleotides that are not encoded on the DNA template strand. This string of adenines is known as the poly(A) tail. With the addition of the cap and tail and completion of splicing, processing of the premRNA is complete. The product is a mature mRNA. 5’ and 3’ untranslated regions (or UTRs) help stabilize the mature RNA and regulate its translation. The mRNAs in bacteria also possess 5’ and 3’ UTRs. Not long after the caps and tails on eukaryotic mRNAs were discovered, evidence began to accumulate that they protect mRNAs from degradation by ribonucleases—enzymes that degrade RNA and enhance the efficiency of translation. RNA processing is the general term for any of the modifications, such as splicing or poly(A) tail addition, needed to convert a primary transcript into a mature RNA. 17.3 An Introduction to Translation To synthesize a protein, the sequences of bases in a messenger RNA molecule is translated into a sequence of amino acids in a polypeptide. Ribosomes are the Site of Protein Synthesis Strong correlation between the number of ribosomes in a given type of cell and the rate at which that cell synthesizes proteins. o Ribosomes are the site of protein synthesis. Translation in Bacteria and Eukaryotes Multiple ribosomes attach to each mRNA, forming a polyribosome. In this way, many copies of a protein can be produced from a single mRNA. Transcription and translation can occur concurrently in bacteria because there is no nuclear envelope to separate the two processes. The situation is different in eukaryotes. In these organisms, primary transcripts are processed in the nucleus to produce a mature mRNA, which is then exported to the cytoplasm. This means that in eukaryotes, transcription and translation are separated in time and space. Once mRNAs are outside the nucleus, ribosomes can attach to them and begin translation. How Does an mRNA Triplet Specify an Amino Acid? When an mRNA interacts with a ribosome, instructions encoded in nucleic acids are translated into a different chemical language—the amino acid sequences found in proteins. One early hypothesis was that mRNA codons and amino acids interact directly. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu Crick proposed an alternative hypothesis. He suggested that some sort of adapter molecule holds amino acids in place while interacting directly and specifically with a codon in mRNA by hydrogen bonding. o Crick was right. 17.4 The Structure and Function of Transfer RNA the adapter molecule was discovered by accident: transfer RNA (tRNA). Amino acids are transferred from tRNAs to proteins. Radioactive amino acids are lost from tRNAs and incorporated into polypeptides synthesized by ribosomes. Amino acids are transferred from the RNA to a growing polypeptide. o tRNAs are Crick’s adapter molecules. What Do tRNAs Look Like? Transfer RNAs serve as chemical gobetweens that allow amino acids to interact with an mRNA template. Two aspects of tRNA’s secondary structure proved especially important. A CCA sequence at the 3’ end of each tRNA molecule offered a site for amino acid attachment, while a triplet on the loop at the other end of the structure could serve as an anticodon. o An anticodon is a set of three ribonucleotides that forms base pairs with the mRNA codon. All the tRNAs in a cell have the same general structure shaped like an upsidedown L. They vary at the anticodon and attached amino acid. The tertiary structure of tRNAs is important because it maintains a precise physical distance between the anticodon and amino acid. This separation is important in positioning the amino acid and the anticodon within the ribosome. How Are Amino Acids Attached to tRNAs? An input of energy, in the form of ATP, is required to attach an amino acid to a tRNA. Enzymes called aminoacyltRNA synthetases catalyze the addition of amino acids to tRNAs—what biologists called “charging” a tRNA. For each of the 20 major amino acids, there is a different aminoacyltRNA synthetase and one or more tRNAs. Each aminoacyltRNA synthetase has a binding site for a particular amino acid and a particular tRNA. Subtle differences in tRNA shape and base sequence allow the enzymes to recognize the correct tRNA for the correct amino acid. The combination of a tRNA molecule covalently linked to an amino acid is called an aminoacyl tRNA. How Many tRNAs Are There? Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu Wobble hypothesis Many amino acids are specified by more than one codon. Codons for the same amino acid tend to have the same nucleotides at the first and second positions but a different nucleotide at the third position. Crick proposed that inside the ribosome, certain bases in the third position of tRNA anticodons can bind to bases in the third position of a codon in a manner that does not match WatsonCrick base pairing. If so, this would allow a limited flexibility, or “wobble,” in the basepairing. o This allows just 40 tRNAs bind to all 61 mRNA codons. 17.5 The Structure and Function of Ribosomes Ribosomes contain many proteins and ribosomal RNAs (rRNAs). Later work showed that ribosomes can be separated into two major substructures, called the large subunit and small subunit. o Each ribosome subunit consists of a complex of RNA molecules and proteins. The small subunit holds the mRNA in place during translation; the large subunit is where peptidebond formation takes place. 3 distinct tRNAs are lined up inside the ribosome. All three are bound to their corresponding mRNA codons. o A site = acceptor or aminoacyl o P site = peptidebond formation o E site = exit The ribosome is a macromolecular machine that synthesizes proteins in a threestep sequence. 1. An aminoacyl tRNA diffuses into the A site; if its anticodon matches a codon in mRNA, it stays in the ribosome. 2. A peptide bond forms between the amino acid held by the aminoacyl tRNA in the A site and the growing polypeptide, which was held by a tRNA in the P site. 3. The ribosome moves down the mRNA by one codon, and all three tRNAs move one position within the ribosome. The tRNA in the E site exits; the tRNA in the P site moves to the E site; and the tRNA in the A site switches to the P site. Initiation Translation The process begins when a section of rRNA in a small ribosomal subunit binds to a complementary sequence on an mRNA. The mRNA region is called the ribosome binding site, or ShineDalgarno sequence. o The site is about six nucleotides upstream from the start codon. o The interactions between the small subunit, the message and the tRNA are mediated by proteins called initiation factors. Initiation factors help in preparing the ribosome for translation, including binding the first aminoacyl tRNA to the ribosome. In bacteria, this initiator tRNA bears a modified form of methionine called Nformylmethionine (abbreviated fmet). Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu In eukaryotes, this initiating tRNA carries a normal methionine. Initiation factors also prevent the small and large subunits of the ribosome from coming together until the initiator tRNA is in place at the AUG start codon, and they help bind the mRNA to the small ribosomal subunit. Translation initiation is a threestep process in bacteria: 1. The mRNA binds to a small ribosomal subunit 2. The initiator aminoacyl tRNA bearing fmet binds to the start codon, and 3. The large ribosomal subunit binds, completing the complex. Elongation: Extending the Polypeptide Elongation proceeds when an aminoacyl tRNA binds to the codon in the A site by complementary basepairing between the anticodon and codon. When both the P site and A site are occupied by tRNAs, the amino acids on the tRNAs are in the ribosome’s active site. o This is where peptidebond formation—the essence of protein synthesis—occurs. Is the Ribosome an Enzyme or a Ribozyme? Active sites consist entirely of ribosomal RNA. Protein synthesis is catalyzed by RNA. The ribosome is a ribozyme (a RNA molecule with catalytic activity)—not a proteinbased enzyme. Moving Down the mRNA Translocation – occurs when proteins called elongation factors help move the ribosome relative to the mRNA so that translation occurs in the 5’ 3’ direction. Translocation is an energydemanding event that requires GTP; Translocation does several things: it moves the uncharged RNA (no longer connected to the amino acid)into the E site; it moves the tRNA containing the growing polypeptide into the P site; and it opens the A site and exposes a new mRNA codon. The empty tRNA that finds itself in the E site is ejected into the cytosol. The three steps in elongation— 1. Arrival of aminoacyl tRNA 2. Peptidebond formation 3. Translocation Terminating Translation When the translocating ribosome reaches one of the stop codons, a protein called a release factor recognizes the stop codon and fills the A site. Release factors do not carry an amino acid o The protein’s active site catalyzes the hydrolysis of the bond that links the tRNA in the P site to the polypeptide chain. Chapter 17 – Transcription, RNA Processing, and Translation MingHan Lu This reaction frees the polypeptide. PostTranslational Modifications Proteins are not fully formed and functional when termination occurs. Proteins go through an extensive series of processing steps called posttranslational modifications. Folding A protein’s function depends on its shape, and that a protein’s shape depends on on how it folds. Although folding can occur spontaneously, it is frequently speeded up by proteins called molecular chaperones. Chemical Modifications Many completed proteins are altered by enzymes that add or remove a phosphate group. Phosphorylation (addition of phosphate) and dephosphorylation (removal of phosphate) can cause major changes in the shape and chemical reactivity of proteins. These changes have a dramatic effect on the protein’s activity—often switching it from an inactive state to an active state, or vice versa.