Protein Synthesis
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Gene$c informa$on is most important because of the proteins that it encodes, in that proteins play most of the func$onal roles in cells. We now turn to the mechanism of protein synthesis, a process called transla'on because the four-­‐le6er alphabet of nucleic acids is translated into the en8rely different twenty-­‐le6er alphabet of proteins. Transla$on is a conceptually more complex process than either replica$on or transcrip$on, both of which take place within the framework of a common base-­‐pairing language. As befits its posi8on linking the nucleic acid and protein languages, the process of protein synthesis cri8cally depends on both nucleic acid and protein factors. Protein synthesis takes place on ribosomes—enormous complexes containing three large RNA molecules and more than 50 proteins. Among the great triumphs in biochemistry in recent years has been the determina$on of the structure of the ribosome so that its func8on can be examined in atomic detail. Ada E. Yonath Nobel Prize (2009) Perhaps the most significant conclusion from these studies is that the ribosome is a ribozyme; that is, the RNA components play the most fundamental roles. These observa8ons strongly support the no8on that life evolved through an RNA world, and the ribosome is a surviving inhabitant of that world. tRNAs and mRNA are also key par8cipants in protein synthesis. The link between amino acids and nucleic acids is made by aminoacyl-­‐tRNA synthetases. By specifically linking a par$cular amino acid to each tRNA, these enzymes translate the gene$c code Although RNA is paramount in the process of transla8on, protein factors also are required for the efficient synthesis of a protein. Protein factors par8cipate in the ini$a$on, elonga$on, and termina$on of protein synthesis. P-­‐loop NTPases of the G-­‐protein family play par8cularly important roles. Recall that these proteins serve as molecular switches as they cycle between a GTP-­‐bound form and a GDP-­‐bound form . This chapter focuses primarily on protein synthesis in prokaryotes because it illustrates many general principles and is well understood. Some dis8nc8ve features of protein synthesis in eukaryotes also are presented. 30.1 Protein Synthesis Requires the Transla$on of Nucleo$de Sequences into Amino Acid Sequences The basics of protein synthesis are the same across all kingdoms of life—evidence that the protein-­‐synthesis system arose very early in evolu$on. An mRNA is decoded in the 5'-­‐to-­‐3' direc8on, one codon at a $me, and the corresponding protein is synthesized in the amino-­‐to-­‐carboxyl direc$on by the sequen$al addi$on of amino acids to the carboxyl end of the growing pep$de chain (Figure 30.1). The amino acids arrive at the growing chain in ac8vated form as aminoacyl-­‐tRNAs, created by joining the carboxyl group of an amino acid to the 3' end of a tRNA molecule. The linking of an amino acid to its corresponding tRNA is catalyzed by an aminoacyl-­‐tRNA synthetase. ATP cleavage drives this ac8va8on reac8on. For each amino acid, there is usually one ac$va$ng enzyme and at least one kind of tRNA. The synthesis of long proteins requires a low error frequency The complexity of transla8on creates a conflict between two requirements: the process must be both accurate and fast enough to meet a cell’s needs. In E. coli, transla8on can take place at a rate of 50 amino acids per second, a truly impressive speed, considering the complexity of the process. How accurate must protein synthesis be? In fact, the observed values of ε are close to 10-­‐4. ~11 kd ~33 kd An error frequency of about 10-­‐4 ~110 kd per amino acid residue was selected in the course of evolu8on to accurately produce proteins consis8ng of as many as 1000 amino acids while maintaining a remarkably rapid rate for protein synthesis. Transfer RNA molecules have a common design The fidelity of protein synthesis requires accurate recogni$on of three-­‐base codons on mRNA. An amino acid cannot itself recognize a codon. Consequently, an amino acid is a6ached to a specific tRNA molecule that can recognize the codon by Watson–Crick base-­‐pairing. Transfer RNA serves as the adapter molecule that binds to a specific codon and brings with it an amino acid for incorpora8on into the polypep$de chain. Consider yeast alanyl-­‐tRNA, so called because it will carry the amino acid alanine. Yeast alanyl-­‐tRNA was the first nucleic acid sequenced. This adapter molecule is a single chain of 76 ribonucleo8des (Figure 30.2). 5' terminus is phosphorylated (pG), whereas the 3' terminus has a free hydroxyl group. The amino acid-­‐a6achment site is the 3'-­‐hydroxyl group of the adenosine residue at the 3' terminus of the molecule. The sequence 5'-­‐IGC-­‐3' in the middle of the molecule is the an$codon, where I is the purine base inosine. Guanine Inosine Adenine It is complementary to 5'-­‐GCC-­‐3', one of the codons for alanine Thousands of tRNA sequences are now known. The striking finding is that all of them can be arranged in a cloverleaf pa6ern in which about half the residues are base-­‐paired (Figure 30.3). Hence, tRNA molecules have many common structural features. All tRNA molecules must be able to interact in nearly the same way with the ribosomes, mRNAs, and protein factors that par9cipate in transla9on. 1. Each is a single chain containing between 73 ~ 93 ribonucleo$des (~25 kd). 2. They contain many unusual bases. Some of these bases are methylated or dimethylated deriva8ves of A, U, C, G Methyla8on imparts a hydrophobic character to some regions of tRNAs, which may be important for their interac8on with synthetases and ribosomal proteins. Other modifica8ons alter codon recogni$on 3. The molecule is L-­‐shaped (Figure 30.4). 4. About half the nucleo8des in tRNAs are base-­‐paired to form double helices. Two apparently con8nuous segments of double helix. These segments are like A-­‐form DNA, as expected for an RNA helix. Five groups of bases are not base-­‐paired in this way: 3' CCA terminal region called the acceptor stem; TψC loop ribothymine-­‐pseudouracil-­‐cytosine; Extra arm contains a variable number of residues; DHU loop contains several dihydrouracil residues; An$codon loop. Most of the bases in the nonhelical regions par8cipate in hydrogen-­‐bonding interac8ons, even if the interac8ons are not like those in Watson–Crick base pairs. The structural diversity generated by this combina8on of helices and loops containing modified bases ensures that the tRNAs can be uniquely dis$nguished, though structurally similar overall. 5. The 5' end of a tRNA is phosphorylated. The 5' terminal residue is usually pG. 6. An ac$vated amino acid is acached to a hydroxyl group of the adenosine residue in the amino acid-­‐acachment site, located at the end of the 3' CCA component of the acceptor stem (Figure 30.6). This single-­‐stranded region can change conforma8on in the course of amino acid ac8va8on and protein synthesis. 7. The an$codon loop, which is present in a loop near the center of the sequence, is at the other end of the L, making accessible the three bases that make up the an8codon. Thus, the architecture of the tRNA molecule is well suited to its role as adaptor: the an8codon is available to interact with an appropriate codon on mRNA while the end that is linked to an ac8vated amino acid is well posi8oned to par8cipate in pep$de-­‐bond forma$on. Some transfer RNA molecules recognize more than one codon because of wobble in base-­‐pairing What are the rules that govern the recogni$on of a codon by the an$codon of a tRNA? A simple hypothesis is that each of the bases of the codon forms a Watson–Crick type of base pair with a complementary base on the an8codon of the tRNA. According to this model, a par'cular an'codon can recognize only one codon. The facts are otherwise. As found experimentally, some tRNA molecules can recognize more than one codon. For example, the yeast alanyl-­‐tRNA binds to three codons: GCU, GCC, GCA. The first two bases are the same, whereas the third is different. Could it be that recogni$on of the third base of a codon is some$mes less discrimina$ng than recogni8on of the other two? The pacern of degeneracy of the gene8c code indicates that it might be so. XYU and XYC always encode the same amino acid; XYA and XYG usually do. Francis Crick surmised from these data that the steric criteria might be less stringent for pairing of the third base than for the other two. Models of various base pairs were built to determine which ones are similar to the standard A Ÿ U and G Ÿ C base pairs with regard to the distance and angle between the glycosidic bonds. Inosine was included in this study because it appeared in several an8codons. With the assump8on of some steric freedom (“wobble”) in the pairing of the third base of the codon, the combina8ons shown in Table 30.2 seemed plausible. The wobble hypothesis is now firmly established. For example, the an8codon is IGC. This tRNA recognizes the codons GCU, GCC, GCA. Recall that, by conven8on, nucleo8de sequences are wricen in the 5’ à 3' direc8on unless otherwise noted. Hence, I (the 5' base of this an$codon) pairs with U, C, or A (the 3' base of the codon), as predicted. Two generaliza$ons concerning the codon–an8codon interac8on can be made: 1. The first two bases of a codon pair in the standard way. Recogni$on is precise. Hence, codons that differ in either of their first two bases must be recognized by different tRNAs. For example, both UUA and CUA encode leucine but are read by different tRNAs. 2. The first base of an an$codon determines whether tRNA reads one, two, or three kinds: C or A (one codon), U or G (two codons), or I (three codons). Thus, part of the degeneracy of the gene8c code arises from imprecision (wobble) in the pairing of the third base of the codon with the first base of the an8codon. We see here a strong reason for the frequent appearance of inosine, one of the unusual nucleosides, in an8codons. Inosine maximizes the number of codons that can be read by a par8cular tRNA molecule. The inosine bases in tRNA are formed by the deamina$on of adenosine aier the synthesis of the primary transcript. Why is wobble tolerated in the third posi$on of the codon but not in the first two? This ques$on is answered by considering the interac$on of the tRNA with the ribosome. As we will see, ribosomes are huge RNA–protein complexes consis8ng of two subunits, the 30S and 50S subunits. The 30S subunit has an RNA molecule, 16S rRNA, that has three universally conserved bases—
adenine 1492, adenine 1493, and guanine 530—
that form hydrogen bonds on the minor-­‐groove side but only with correctly formed base pairs of the codon–an8codon duplex (Figure 30.7) These interac8ons serve to check whether Watson–Crick base pairs are present in the first two posi$ons of the codon–an8codon duplex. No such inspec$on device is present for the third posi$on; so more-­‐varied base pairs are tolerated. Thus, the ribosome plays an ac've role in decoding the codon– an'codon interac'ons. 30.2 Aminoacyl Transfer RNA Synthetases Read the Gene$c Code Before codon and an8codon meet, the amino acids required for protein synthesis must first be a;ached to specific tRNA molecules. The linkage of an amino acid to a tRNA is crucial for two reasons. First, the a6achment of a given amino acid to a par$cular tRNA establishes the gene$c code. When an amino acid has been linked to a tRNA, it will be incorporated into a growing polypep8de chain at a posi8on dictated by the an$codon of the tRNA. Second, because the forma8on of a pep$de bond between free amino acids is not thermodynamically favorable, the amino acid must first be ac$vated in order for protein synthesis to proceed. The ac8vated intermediates in protein synthesis are amino acid esters, in which the carboxyl group of an amino acid is linked to either the 2'-­‐ or the 3'-­‐hydroxyl group of the ribose unit at the 3' end of tRNA. An amino acid ester of tRNA is called an aminoacyl-­‐tRNA (see Figure 30.6). For a specific amino acid acached to its cognate tRNA—for instance, threonine—the charged tRNA is designated Thr-­‐tRNAThr. Amino acids are first ac$vated by Adenyla$on The ac8va8on reac8on is catalyzed by specific aminoacyl-­‐tRNA synthetases, which are also called ac$va$ng enzymes. The first step is the forma8on of an aminoacyl adenylate from an amino acid and ATP. Amino acid + ATP  aminoacyl-­‐AMP + PPi This ac8vated species is a mixed anhydride in which the carboxyl group of the amino acid is linked to the phosphoryl group of AMP; hence, it is also known as aminoacyl-­‐AMP. The next step is the transfer of the aminoacyl group of aminoacyl-­‐AMP to a par8cular tRNA molecule to form aminoacyl-­‐tRNA. Aminoacyl-­‐AMP + tRNA à aminoacyl-­‐tRNA + AMP The sum of these ac8va8on and transfer steps is Amino acid + ATP + tRNA à aminoacyl-­‐tRNA + AMP+ PPi The ΔG°’ of this reac$on is close to 0, because the free energy of hydrolysis of the ester bond of aminoacyl-­‐tRNA is similar to that for the hydrolysis of ATP to AMP and PPi. The reac$on is driven by the hydrolysis of pyrophosphate. The sum of these three reac8ons is highly exergonic: Amino acid + ATP + tRNA + H2Oà aminoacyl-­‐tRNA + AMP+ 2Pi Thus, the equivalent of two molecules of ATP is consumed in the synthesis of each aminoacyl-­‐tRNA. One of them is consumed in forming the ester linkage of aminoacyl-­‐tRNA, whereas the other is consumed in driving the reac$on forward. Aminoacyl-­‐tRNA synthetases have highly discrimina$ng amino acid ac$va$on sites Each aminoacyl-­‐tRNA synthetase is highly specific for a given amino acid. Indeed, a synthetase will incorporate the incorrect amino acid only once in 104 or 105 cataly8c reac8ons. How is this level of specificity achieved? Let us consider the challenge faced by threonyl-­‐tRNA synthetase. Threonine is par8cularly similar to two other amino acids—namely, valine and serine. How can the threonyl-­‐tRNA synthetase avoid coupling these incorrect amino acids to threonyl-­‐tRNA? The structure of the amino acid-­‐binding site of threonyl-­‐tRNA synthetase reveals how valine is avoided (Figure 30.8). The synthetase contains a zinc ion, bound to the enzyme by two his$dine residues and on cysteine residue. The remaining coordina8on sites are available for substrate binding. Threonine coordinates to the zinc ion through its amino group and its side-­‐chain hydroxyl group. The side-­‐
chain hydroxyl group is further recognized by an aspartate residue that hydrogen bonds to it. The methyl group present in valine in place of this hydroxyl group cannot par$cipate in these interac$ons; it is excluded from this ac8ve site and, hence, does not become adenylated and transferred to threonyl-­‐tRNA (abbreviated tRNAThr). The use of a zinc ion appears to be unique to threonyl-­‐tRNA synthetase; other aminoacyl-­‐
tRNA synthetases have different strategies for recognizing their cognate amino acids. The zinc site is less able to discriminate against serine because this amino acid does have a hydroxyl group that can bind to the zinc ion. Indeed, with only this mechanism available, threonyl-­‐tRNA synthetase does mistakenly couple serine to threonyl-­‐tRNA at a rate 10-­‐2 to 10-­‐3 $mes that for threonine. Proofreading by aminoacyl-­‐tRNA synthetases increases the fidelity of protein synthesis Threonyl-­‐tRNA synthetase can be incubated with tRNAThr that has been covalently linked with serine (Ser-­‐tRNAThr); the tRNA has been “mischarged.” A rapid hydrolysis of the aminoacyl-­‐tRNA forms serine and free tRNA. In contrast, incuba8on with correctly charged Thr-­‐tRNAThr results in no reac8on. Thus, threonyl-­‐ tRNA synthetase contains an addi8onal func8onal site that hydrolyzes Ser-­‐tRNAThr but not Thr-­‐tRNAThr. This edi$ng site provides an opportunity for the synthetase to correct its mistakes and improve its fidelity to less than one mistake in 104. This edi8ng site readily accepts and cleaves Ser-­‐tRNAThr but does not cleave Thr-­‐tRNAThr. The discrimina$on of serine from threonine is easy because threonine contains an extra methyl group; a site that conforms to the structure of serine will sterically exclude threonine. Most aminoacyl-­‐tRNA synthetases contain edi$ng sites in addi8on to ac8va8on sites. These complementary pairs of sites func8on as a double sieve to ensure very high fidelity. In general, the acyla$on site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydroly$c site cleaves ac$vated species that are smaller than the correct one. The structure of the complex between threonyl-­‐tRNA synthetase and its substrate reveals that the aminoacylated CCA can swing out of the ac$va$on site and into the edi$ng site (Figure 30.10). Thus, the aminoacyl-­‐tRNA can be edited without dissocia8ng from the synthetase. This proofreading, which depends on the conforma8onal flexibility of a short stretch of polynucleo8de sequence, is en8rely analogous to that of DNA polymerase (Sec8on 28.1). In both cases, edi'ng without dissocia'on significantly improves fidelity with only modest costs in 'me and energy. Synthetases recognize various features of transfer RNA molecules How do synthetases choose their tRNA partners? This enormously important step is the point at which “transla8on” takes place—at which the correla8on between the amino acid and the nucleic acid worlds is made. In a sense, aminoacyl-­‐tRNA synthetases are the only molecules in biology that “know” the gene8c code. Their precise recogni8on of tRNAs is as important for high-­‐fidelity protein synthesis as is the accurate selec8on of amino acids. In general, tRNA recogni8on by the synthetase is different for each synthetase and tRNA pair. Consequently, generali$es are difficult to make. Some synthetases recognize their tRNA partners primarily on the basis of their an$codons, although they may also recognize other aspects of tRNA structure that vary among different tRNAs. The most direct evidence comes from crystallographic studies of complexes formed between synthetases and their cognate tRNAs. Consider, for example, the structure of the complex between threonyl-­‐tRNA synthetase and tRNAThr (Figure 30.11). As expected, the CCA arm extends into the zinc-­‐
containing ac8va8on site, where it is well posi8oned to accept threonine from threonyl adenylate. The enzyme interacts extensively not only with the acceptor stem of the tRNA, but also with the an$codon loop. Each base within the sequence CGU of the an$codon par$cipates in hydrogen bonds with the enzyme; those with the second two bases (G and U) appear to be more important because the synthetase interacts just as efficiently with the an8codons GGU and UGU. Although interac$ons between the enzyme and the an$codon are oien crucial for correct recogni8on, Figure 30.12 shows that many aspects of tRNA molecules are recognized by synthetases. Note that many of the recogni8on sites are loops rich in unusual bases that can provide structural iden$fiers. Figure 30.12 Recogni$on Sites on tRNA Circles represent nucleo8des, and the sizes of the circles are propor8onal to the frequency with which they are used as recogni8on sites by aminoacyl-­‐tRNA synthetases. Aminoacyl-­‐tRNA synthetases can be divided into two classes At least one aminoacyl-­‐tRNA synthetase exists for each amino acid. The determina8on of the three-­‐dimensional structures of several synthetases followed by more-­‐refined sequence comparisons revealed that different synthetases are, in fact, related. Specifically, synthetases fall into two classes, termed class I and class II, each of which includes enzymes specific for 10 of the 20 amino acids (Table 30.3). Intriguingly, synthetases from the two classes bind to different faces of the tRNA molecule (Figure 30.13). Note that the CCA arm of the tRNA is turned toward the viewer The CCA arm of tRNA adopts different conforma$ons to accommodate these interac8ons; the arm is in the helical conforma8on observed for free tRNA for class II enzymes and in a hairpin conforma8on for class I enzymes. These two classes also differ in other ways. 1.  Class I enzymes acylate the 2’-­‐hydroxyl group of the terminal adenosine whereas class II enzymes acylate the 3’-­‐hydroxyl group 2. The two classes bind ATP in different conforma$ons. 3. Most class I enzymes are monomeric, whereas most class II enzymes are dimeric. Why did two dis$nct classes of aminoacyl-­‐tRNA synthetases evolve? The observa8on that the two classes bind to dis8nct faces of tRNA suggests a possibility. Recogni$on sites on both faces of tRNA may have been required to allow the recogni$on of 20 different tRNAs. 30.3 The Ribosome Is the Site of Protein Synthesis We turn now to ribosomes, the molecular machines that coordinate the interplay of charged tRNAs, mRNA, and proteins that leads to protein synthesis. An E. coli ribosome is a ribonucleoprotein assembly with a mass of about 2500 kd, a diameter of approximately 250 Å, and a sedimenta8on coefficient of 70S. The 20,000 ribosomes in a bacterial cell cons8tute nearly a fourth of its mass. A ribosome can be dissociated into a large subunit (50S) and a small subunit (30S). These subunits can be further split into their cons8tuent proteins and RNAs. 30S contains 21 different proteins (referred to as S1 through S21) and a 16S RNA molecule. 50S contains 34 different proteins (L1 through L34) and two RNA, a 23S and a 5S species. Both the 30S and the 50S subunits can be recons$tuted in vitro from their cons8tuent proteins and RNA, as was first achieved by Masayasu Nomura in 1968. This recons9tu9on is an outstanding example that supramolecular complexes can form spontaneously from their macromolecular cons9tuents. Astounding progress on the structure of the ribosome has been made by x-­‐ray crystallographic methods, aier the pioneering work by Ada Yonath (Nobel prize 2009). The structures of both the 30S and the 50S subunits as well as the complete 70S ribosome have been determined at or close to atomic resolu$on (Figure 30.14). The determina8on of the structure of the 70S ribosome requires the posi8oning of more than 100,000 atoms. Ribosomal RNAs (5S, 16S, and 23S rRNA) play a central role in protein synthesis 3 RNAs present: 5S, 16S, 23S. These molecules fold into structures that allow them to form internal base pairs (base-­‐pairing). conserved sequences. For instance, the 16S RNA of one species may have a G–C base pair, whereas another may have an A–U base pair, but the loca'on of the base pair is the same in both molecules. The striking finding is that across all species ribosomal RNAs (rRNAs) are folded into defined structures that have many short duplex regions. 16S Ribosomal RNA folding pa6ern. (A)  Secondary structure of 16S (B)  Ter8ary structure