Protein Synthesis & Genetic Code | Harper's Biochemistry PDF

C H A P T E R Protein Synthesis & the Genetic Code P. Anthony Weil, PhD 37 OBJ EC T IVES Understand that the genetic code is a three-letter nucleotide code, w...

C H A P T E R Protein Synthesis & the Genetic Code P. Anthony Weil, PhD 37 OBJ EC T IVES Understand that the genetic code is a three-letter nucleotide code, which is contained within the linear array of the exon DNA (composed of triplets After studying this chapter, of A, G, C, and T) of protein coding genes, and that this three-letter code is you should be able to: translated into mRNA (composed of triplets of A, G, C, and U) to specify the linear order of amino acid addition during protein synthesis via the process of translation. Appreciate that the universal genetic code is degenerate, unambiguous, nonoverlapping, and punctuation free. Explain that the genetic code is composed of 64 codons, 61 of which encode amino acids while 3 induce the termination of protein synthesis. Describe how the transfer RNAs (tRNAs) serve as the ultimate informational agents that decode the genetic code of messenger RNAs (mRNAs). Understand the mechanism of the energy-intensive process of protein synthesis that involves the steps of initiation, elongation, and termination, occurs on RNA-protein complexes termed ribosomes. Appreciate that protein synthesis, like DNA replication and transcription, is precisely controlled through the action of multiple accessory factors that are responsive to multiple extra- and intracellular regulatory signaling inputs. BIOMEDICAL IMPORTANCE GENETIC INFORMATION FLOWS The letters A, G, T, and C correspond to the nucleotides found FROM DNA TO RNA TO PROTEIN in DNA. Within the protein-coding genes, these nucleotides The genetic information within the nucleotide sequence of are organized into three-letter code words called codons, and DNA is transcribed in the nucleus into the specific nucleotide the collection of these codons, once transcribed into mRNA, sequence of an mRNA molecule. The sequence of nucleotides make up the genetic code. It was impossible to understand in the RNA transcript is complementary to the nucleotide protein synthesis—or to explain the molecular effects of gene sequence of the template strand of its gene in accordance with mutations—before the genetic code was deciphered. The code Watson-Crick base-pairing rules. Several different classes of provides a foundation for explaining the way in which protein RNA combine to direct the synthesis of proteins. defects may cause genetic disease and for the diagnosis and In prokaryotes there is a linear correspondence between possible treatment of these disorders. In addition, the patho- the gene, the messenger RNA transcribed from the gene, and physiology of many viral infections is related to the ability of the polypeptide product. The situation is more complicated in these infectious agents to disrupt host cell protein synthesis. higher eukaryotic cells where the primary transcript is much Many antibacterial drugs are effective because they selectively larger than the mature mRNA. The large mRNA precursors disrupt protein synthesis in the invading bacterial cell but do contain coding regions (exons) that will form the mature not affect protein synthesis in eukaryotic cells. mRNA and long intervening sequences (introns) that separate 404  CHAPTER 37 Protein Synthesis & the Genetic Code 405 the exons. The mRNA is processed within the nucleus, and TABLE 37–1 The Genetic Codea (Codon Assignments in the introns, which typically make up much more of this RNA Mammalian Messenger RNAs) than the exons, are removed. Exons are spliced together to Second Nucleotide form mature mRNAs, which are transported to the cytoplasm, First Third where they are translated into protein (see Chapter 36). Nucleotide U C A G Nucleotide The cell must possess the machinery necessary to trans- U Phe Ser Tyr Cys U late information accurately and efficiently from the nucleotide sequence of an mRNA into the sequence of amino acids of the Phe Ser Tyr Cys C corresponding specific protein. Clarification of our under- Leu Ser Term Termb A standing of this process, which is termed translation, awaited Leu Ser Term Trp G deciphering of the genetic code. It was realized early that mRNA molecules themselves have no affinity for amino acids and, C Leu Pro His Arg U therefore, that the translation of the information in the mRNA Leu Pro His Arg C nucleotide sequence into the amino acid sequence of a protein Leu Pro Gln Arg A requires an intermediate adapter molecule. This adapter mol- ecule must recognize a specific nucleotide sequence on the one Leu Pro Gln Arg G hand as well as a specific amino acid on the other. With such an A Ile Thr Asn Ser U adapter molecule, the cell can direct a specific amino acid into Ile Thr Asn Ser C the proper sequential position of a protein during its synthesis as dictated by the nucleotide sequence of the specific mRNA. Ilea Thr Lys Argb A The functional groups of the amino acids do not themselves Met Thr Lys Argb G actually come into contact with the mRNA template. G Val Ala Asp Gly U Val Ala Asp Gly C THE NUCLEOTIDE SEQUENCE OF Val Ala Glu Gly A AN mRNA MOLECULE CONTAINS Val Ala Glu Gly G A SERIES OF CODONS THAT SPECIFY THE AMINO ACID a The terms first, second, and third nucleotide refer to the individual nucleotides of a triplet codon read 5′-3′, left to right. A, adenine nucleotide; C, cytosine nucleotide; G, SEQUENCE OF THE ENCODED guanine nucleotide; Term, chain terminator codon; U, uridine nucleotide. AUG, which codes for Met, serves as the initiator codon in mammalian cells and also encodes for inter- PROTEIN nal methionines in a protein. (Abbreviations of amino acids are explained in Chapter 3.) b In mammalian mitochondria, AUA codes for Met and UGA for Trp, and AGA and AGG Twenty different amino acids are required for the synthe- serve as chain terminators. sis of the cellular complement of proteins; thus, there must be at least 20 distinct codons that make up the genetic code. signals by specifying where the polymerization of amino acids Since there are only four different nucleotides in mRNA, each into a protein molecule is to stop. The remaining 61 codons code codon must consist of more than a single purine or pyrimidine for the 20 naturally occurring amino acids (see Table 37–1). nucleotide. Codons consisting of two nucleotides each could Thus, there is “degeneracy” in the genetic code—that is, multi- provide for only 16 (ie, 42) distinct codons, whereas codons of ple codons decode the same amino acid. Some amino acids are three nucleotides could provide 64 (43) specific codons. encoded by several codons; for example, six different codons, It is now known that each codon consists of a sequence of UCU, UCC, UCA, UCG, AGU, and AGC all specify serine. three nucleotides; that is, it is a triplet code (Table 37–1). The Other amino acids, such as methionine and tryptophan, have initial deciphering of the genetic code depended heavily on a single codon. In general, the third nucleotide in a codon is in vitro synthesis of nucleotide polymers, particularly triplets less important than the first two in determining the specific in repeated sequence. These synthetic triplet ribonucleotides amino acid to be incorporated, and this accounts for most of were used as mRNAs to program protein synthesis in the test the degeneracy of the code. However, for any specific codon, tube, which allowed investigators to deduce the genetic code. only a single amino acid is specified; with rare exceptions, the genetic code is unambiguous—that is, given a specific codon, only a single amino acid is indicated. The distinction between THE GENETIC CODE IS ambiguity and degeneracy is an important concept. DEGENERATE, UNAMBIGUOUS, The unambiguous but degenerate code can be explained NONOVERLAPPING, WITHOUT in molecular terms. The recognition of specific codons in the mRNA by the tRNA adapter molecules is dependent on the PUNCTUATION, & UNIVERSAL tRNA anticodon region and specific base-pairing rules that Three of the 64 possible codons do not code for specific amino dictate tRNA–mRNA codon binding. Each tRNA molecule acids; these have been termed nonsense codons. Nonsense contains a specific sequence, complementary to a codon, which codons are utilized in the cell as translation termination is termed its anticodon. For a given codon in the mRNA, 406 SECTION VII Structure, Function, & Replication of Informational Macromolecules only a single species of tRNA molecule possesses the proper TABLE 37–2 Features of the Genetic Code anticodon. Since each tRNA molecule can be charged with Degenerate only one specific amino acid, each codon therefore specifies Unambiguous only one amino acid. However, some tRNA molecules can uti- Nonoverlapping lize the anticodon to recognize more than one codon. With Not punctuated Universal few exceptions, given a specific codon, only a specific amino acid will be incorporated—although, given a specific amino acid, more than one codon may be used. A summary of the main features of the genetic code are listed As discussed in the following section, the reading of the in Table 37–2. genetic code during the process of protein synthesis does not involve any overlap of codons. Thus, the genetic code is non- overlapping. Furthermore, once the reading is commenced AT LEAST ONE SPECIES OF at a specific start codon, there is no punctuation between tRNA EXISTS FOR EACH OF codons, and the message is read in a continuing sequence of nucleotide triplets until a translation stop codon is reached. THE 20 AMINO ACIDS Until recently, the genetic code was thought to be univer- tRNA molecules have extraordinarily similar functions and sal. It has now been shown that the set of tRNA molecules in three-dimensional structures. The adapter function of the mitochondria (which contain their own separate and distinct tRNA molecules requires the charging of each specific tRNA translation machinery) from lower and higher eukaryotes, with its specific amino acid. Since there is no affinity of nucleic including humans, reads four codons differently from the acids for specific functional groups of amino acids, this rec- tRNA molecules in the cytoplasm of even the same cells. As ognition must be carried out by a protein molecule capable noted in a footnote to Table 37–1, in mammalian mitochon- of recognizing both a specific tRNA molecule and a specific dria the codon AUA is read as Met, and UGA codes for Trp. In amino acid. At least 20 specific enzymes are required for these addition, in mitochondria, the codons AGA and AGG are read specific recognition functions and for the proper attachment as stop or chain terminator codons rather than as Arg. As a of the 20 amino acids to specific tRNA molecules. This energy result of these organelle-specific changes in genetic code, mito- requiring process of recognition and attachment, tRNA chondria require only 22 tRNA molecules (see Figure 35–8 amino acid charging, proceeds in two steps and is catalyzed for the location of these genes in mtDNA) to read their genetic by one enzyme for each of the 20 amino acids. These enzymes code, whereas the cytoplasmic translation system possesses a are termed aminoacyl-tRNA synthetases. They form an full complement of 31 tRNA species. These exceptions noted, activated intermediate of aminoacyl-AMP–enzyme complex the genetic code is universal. The frequency of use of each (Figure 37–1). The specific aminoacyl-AMP–enzyme com- amino acid codon varies considerably between species and plex then recognizes a specific tRNA to which it attaches the among different tissues within a species. The specific tRNA aminoacyl moiety at the 3′-hydroxyl adenosyl terminal. The levels generally mirror these codon usage biases. Thus, a particu- charging reactions have an error rate of less than 10−4 and so lar abundantly used codon is decoded by a similarly abundant- are quite accurate. The amino acid remains attached to its specific tRNA which recognizes that particular codon. Tables of specific tRNA in an ester linkage until it is incorporated at a codon usage are quite accurate now that many genomes have specific position during the synthesis of a polypeptide on the been sequenced and such information is vital for large-scale ribosome. production of proteins for therapeutic purposes (ie, insulin, The regions of the tRNA molecule referred to in Chapter 34 erythropoietin). Such proteins are often produced in nonhu- (and illustrated in Figure 34–11) now become important. The man cells using recombinant DNA technology (see Chapter 39). ribothymidine pseudouridine cytidine (TψC) arm is involved ATP PPi AMP + Enz O O HOOC HC R Enz Adenosine O P O C CH R H2N OH NH2 Enzyme (Enz) Enz AMP-aa tRNA tRNA-aa (Activated amino acid) Aminoacyl- Amino acid (aa) tRNA synthetase Aminoacyl-AMP-enzyme Aminoacyl-tRNA complex FIGURE 37–1 Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme aminoacyl-tRNA synthetase, results in the for- mation of aminoacyl-tRNA. The first reaction involves the formation of an AMP-amino acid–enzyme complex. This activated amino acid is next transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the latter can be reutilized. The charging reactions have an error rate (ie, esterifying the incorrect amino acid on tRNAXXX) of less than one mischarging event out of 104 amino acid charging events.  CHAPTER 37 Protein Synthesis & the Genetic Code 407 in binding of the aminoacyl-tRNA to the ribosomal surface at MUTATIONS RESULT WHEN the site of protein synthesis. The tRNA dihydrouridine (D) arm is one of the sites important for the proper recognition of a CHANGES OCCUR IN THE given tRNA species by its proper aminoacyl-tRNA synthetase. NUCLEOTIDE SEQUENCE The tRNA acceptor arm, located at the 3′-hydroxyl adenosyl Although the initial change may not occur in the template terminal, is the site of attachment of the specific amino acid. strand of the double-stranded DNA molecule for that gene, The anticodon region (arm) consists of seven nucleotides, after replication, daughter DNA molecules with mutations in and it recognizes the three-letter codon in mRNA (Figure 37–2). the template strand will segregate and appear in the popula- The sequence read from the 3′ to 5′ direction in that anticodon tion of organisms. loop consists of a variable base (N)–modified purine (Pu*)–XYZ (the anticodon)–pyrimidine (Py)–pyrimidine (Py)-5′. Note that this direction of reading the anticodon is 3′–5′, whereas the Some Mutations Occur by Base genetic code in Table 37–1 is read 5′–3′, since the codon and the Substitution anticodon loop of the mRNA and tRNA molecules, respectively, Single-base changes (point mutations) may be transitions or are antiparallel in their complementarity just like all other inter- transversions. In the former, a given pyrimidine is changed to molecular interactions between nucleic acid strands. the other pyrimidine or a given purine is changed to the other The degeneracy of the genetic code resides mostly in the last purine. Transversions are changes from a purine to either of nucleotide of the codon triplet, suggesting that the base pairing the two pyrimidines or the change of a pyrimidine into either between this last nucleotide and the corresponding nucleotide of the two purines, as shown in Figure 37–3. of the anticodon is not strictly by the Watson-Crick rule. This When the DNA nucleotide sequence of a protein-coding is called wobble; the pairing of the codon and anticodon can gene containing the mutation is transcribed into an mRNA “wobble” at this specific nucleotide-to-nucleotide pairing site. molecule, then the RNA molecule will of course possess the For example, the two codons for arginine, AGA and AGG, can base change at the corresponding location. bind to the same anticodon having an uracil at its 5′ end (UCU). Single-base changes in the mRNA may have one of several Similarly, three codons for glycine—GGU, GGC, and GGA— effects when translated into protein: can form a base pair from one anticodon, 3′ CCI 5′, because inosine (I) can base pair with U, C, and A. Inosine is generated 1. There may be no detectable effect because of the degeneracy by deamination of adenine (see Figure 33–2 for structure). of the code; such mutations are often referred to as silent mutations. This would be most likely if the changed base in the mRNA molecule were to be at the third nucleotide of a codon. Because of wobble, the translation of a codon is least sensitive to a change at the third position. 2. A missense effect will occur when a different amino acid is incorporated at the corresponding site in the protein mol- ecule. This mistaken amino acid—or missense, depending on its location in the specific protein—might be accept- able, partially acceptable, or unacceptable to the function of that protein molecule. From a careful examination of the genetic code, one can conclude that most single-base changes would result in the replacement of one amino acid by another with rather similar functional groups. This is an effective genetic “buffering” mechanism to avoid drastic change in the physical properties of a protein molecule. If an acceptable missense effect occurs, the resulting protein molecule may not be distinguishable from the normal one. T C T A A T FIGURE 37–2 Recognition of the codon by the anticodon. One of the codons for phenylalanine is UUU. tRNA charged with phenylalanine (Phe) has the complementary sequence AAA; hence, A G C G G C it forms a base-pair complex with the codon. The anticodon region (arm) typically consists of a sequence of seven nucleotides: variable Transitions Transversions (N), modified purine (Pu*), X, Y, Z (here, A A A), and two pyrimidines (Py) in the 3′ to 5′ direction. Note the antiparallel mode of interaction FIGURE 37–3 Diagrammatic representation of transition between mRNA and tRNA. and transversion mutations. 408 SECTION VII Structure, Function, & Replication of Informational Macromolecules A partially acceptable missense will result in a protein mol- polypeptide both garbled and prematurely terminated (Example 3, ecule with partial but abnormal function. If an unaccept- Figure 37–5). able missense effect occurs, then the protein molecule will If three nucleotides or a multiple of three nucleotides are not be capable of functioning normally. deleted from a coding region, translation of the corresponding mRNA will generate a protein that is missing the correspond- 3. A nonsense codon may appear that would then result in the ing number of amino acids (Example 2, Figure 37–5). Because premature termination of translation and the production the reading frame is a triplet, the reading phase will not be of only a fragment of the intended protein molecule. The disturbed for those codons distal to the deletion. If, however, probability is high that a prematurely terminated protein deletion of one or two nucleotides occurs just prior to or molecule or peptide fragment will not function in its nor- within the normal termination codon (nonsense codon), the mal role. Examples of the different types of mutations, and reading of the normal termination signal is disturbed. Such a their effects on the coding potential of mRNA are presented deletion might result in reading through the now “mutated” in Figures 37–4 and 37–5. termination signal until another nonsense codon is encoun- tered (not shown here). Frameshift Mutations Result From Insertions of one or two or nonmultiples of three nucle- otides into a gene result in an mRNA in which the reading Deletion or Insertion of Nucleotides in frame is distorted on translation, and the same effects that DNA That Generates Altered mRNAs occur with deletions are reflected in the mRNA translation. The deletion of a single nucleotide from the coding strand of This may result in garbled amino acid sequences distal to a gene results in an altered reading frame in the mRNA. The the insertion and the generation of a nonsense codon at, or machinery translating the mRNA does not recognize that a distal to the insertion, or perhaps reading through the normal base was missing, since there is no punctuation in the reading termination codon. Following a deletion in a gene, an inser- of codons. Thus, a major alteration in the sequence of polym- tion (or vice versa) can reestablish the proper reading frame erized amino acids, as depicted in Example 1, Figure 37–5, (Example 4, Figure 37–5). The corresponding mRNA, when results. Altering the reading frame results in a garbled trans- translated, would contain a garbled amino acid sequence lation of the mRNA distal to the single nucleotide deletion. between the insertion and deletion. Beyond the reestablish- Not only is the sequence of amino acids distal to this dele- ment of the reading frame, the amino acid sequence would tion garbled, but reading of the message can also result in the be correct. One can imagine that different combinations of appearance of a nonsense codon and thus the production of a insertions or deletions (ie, indels), or of both, would result in FIGURE 37–4 Examples of three types of missense mutations resulting in abnormal hemoglobin chains. The amino acid altera- tions and possible alterations in the respective codons are indicated. The hemoglobin Hikari β-chain mutation has apparently normal physi- ologic properties but is electrophoretically altered. Hemoglobin S has a β-chain mutation and partial function; hemoglobin S binds oxygen but precipitates when deoxygenated; this causes red blood cells to sickle, and represents the cellular and molecular basis of sickle cell disease (see Figure 6–13). Hemoglobin M Boston, an α-chain mutation, permits the oxidation of the heme ferrous iron to the ferric state and so will not bind oxygen at all.  CHAPTER 37 Protein Synthesis & the Genetic Code 409 Normal Wild type mRNA 5'... UAG UUUG AUG GCC UCU UGC AAA GGC UAU AGU AGU UAG... 3' Polypeptide Met Ala Ser Cys Lys Gly Tyr Ser Ser STOP Example 1 Deletion (–1) –1 U mRNA 5'... UAG UUUG AUG GCC CUU GCA AAG GCU AUA GUA GUU AG... 3' Polypeptide Met Ala Leu Ala Lys Ala Thr Val Val Ser Garbled Example 2 Deletion (–3) –3 UGC mRNA 5'... UAG UUUG AUG GCC UCU AAA GGC UAU AGU AGU UAG... 3' Polypeptide Met Ala Ser Lys Gly Try Ser Ser STOP Example 3 Insertion (+1) +1 C mRNA 5'... UAG UUUG AUG GCC CUC UUG CAA AGG CUA UAG UAG UUAG... 3' Polypeptide Met Ala Leu Leu Gln Arg Leu STOP Garbled Example 4 Insertion (+1) Deletion (–1) +1 U –1 C mRNA 5'... UAG UUUG AUG GCC UCU UUG CAA AGG UAU AGU AGU UAG... 3' Polypeptide Met Ala Ser Leu Gln Arg Tyr Ser Ser STOP Garbled FIGURE 37–5 Examples of the effects of deletions and insertions in a gene on the sequence of the mRNA transcript and of the polypeptide chain translated therefrom. The arrows indicate the sites of deletions or insertions, and the numbers in the ovals indicate the number of nucleotide residues deleted or inserted. Colored type indicates the correct amino acids in the correct order. formation of a protein wherein a portion is abnormal, but this formed as a result of alterations in their anticodon regions, portion is surrounded by the normal amino acid sequences. are capable of suppressing certain missense mutations, non- Such phenomena have been demonstrated convincingly in a sense mutations, and frameshift mutations. However, since number of human diseases. the suppressor tRNA molecules are not capable of distinguish- ing between a normal codon and one resulting from a gene Suppressor Mutations Can Counteract mutation, their presence in the cell usually results in decreased viability. For instance, the nonsense suppressor tRNA mol- Some of the Effects of Missense, ecules can suppress the normal termination signals to allow Nonsense, & Frameshift Mutations a read-through when it is not desirable. Frameshift suppres- The discussion of the altered protein products of gene muta- sor tRNA molecules may read a normal codon plus a compo- tions is based on the presence of normally functioning tRNA nent of a juxtaposed codon to provide a frameshift, also when molecules. However, in prokaryotic and lower eukaryotic it is not desirable. Suppressor tRNA molecules may exist in organisms, abnormally functioning tRNA molecules have mammalian cells, since read-through of translation has on been discovered that are themselves the results of mutations. occasion been observed. In the laboratory context, such sup- Some of these abnormal tRNA molecules are capable of bind- pressor tRNAs, coupled with mutated variants of aminoacyl- ing to and decoding altered codons, thereby suppressing the tRNA synthetases, can be utilized to incorporate unnatural effects of mutations in distinct mutated mRNA-encoding amino acids into defined locations within altered genes that structural genes. These suppressor tRNA molecules, usually carry engineered nonsense mutations. The resulting labeled 410 SECTION VII Structure, Function, & Replication of Informational Macromolecules proteins can be used for in vivo and in vitro cross-linking and factors, eIF-3, eIF-1, and eIF-1A, bind to the newly dissoci- biophysical studies. This new tool adds significantly to biolo- ated 40S ribosomal subunit. Binding of these three eIFs delay gists interested in studying the mechanisms of a wide range of reassociation of the 40S subunit with the 60S subunit, allow- biologic processes. ing other translation initiation factors to associate with the 40S subunit. LIKE TRANSCRIPTION, PROTEIN Formation of the 43S Preinitiation Complex SYNTHESIS CAN BE DESCRIBED The first step of translation initiation involves the binding of IN THREE PHASES: INITIATION, GTP by eIF-2. This binary complex then binds to methionyl ELONGATION, & TERMINATION tRNAi, a tRNA specifically involved in binding to the initiation codon AUG. It is important to note that there are two tRNAs for The general structural characteristics of ribosomes are discussed methionine. One specifies methionine for the initiator codon, in Chapter 34. These particulate entities serve as the machinery the other for internal methionines. Each has a unique nucleotide on which the mRNA nucleotide sequence is translated into the sequence; both are aminoacylated by the same methionyl-tRNA sequence of amino acids of the specified protein. The translation synthetase. The GTP-eIF-2-tRNAi ternary complex binds to the of the mRNA commences near its 5′ end with the formation of 40S ribosomal subunit to form the 43S preinitiation complex. the corresponding amino terminus of the protein molecule. The The ternary complex–40S subunit complex is stabilized by eIF-3 message is decoded from 5′ to 3′, concluding with the formation and eIF-1A and the subsequent binding of eIF5. of the carboxyl terminus of the protein. Again, the concept of eIF-2 is one of two control points for protein synthesis ini- polarity is apparent. As described in Chapter 36, the transcrip- tiation in eukaryotic cells. eIF-2 consists of α, β, and γ subunits. tion of a gene into the corresponding mRNA or its precursor eIF-2α is phosphorylated (on serine 51) by at least four dif- first forms the 5′ end of the RNA molecule. In prokaryotes, this ferent protein kinases (HCR, PKR, PERK, and GCN2) that allows for the beginning of mRNA translation before the tran- are activated when a cell is under stress and when the energy scription of the gene is completed. In eukaryotic organisms, the expenditure required for protein synthesis would be deleteri- process of transcription is a nuclear one, while mRNA trans- ous. Such conditions include amino acid or glucose starva- lation occurs in the cytoplasm, precluding simultaneous tran- tion, virus infection, intracellular presence of large quantities scription and translation in eukaryotic organisms and enabling of misfolded proteins (endoplasmic reticulum [ER] stress), the processing necessary to generate mature mRNA from the serum deprivation (for cells in culture), hyperosmolality, and primary transcript. heat shock. PKR is particularly interesting in this regard. This kinase is activated by viruses and provides a host defense mech- Initiation Involves Several Protein-RNA anism that decreases protein synthesis, including viral protein Complexes synthesis, thereby inhibiting viral replication. Phosphorylated Initiation of eukaryotic protein synthesis requires that an eIF-2α binds tightly to and inactivates the GTP–GDP recycling mRNA molecule be selected for translation by a ribosome protein eIF-2B, thus preventing formation of the 43S preinitia- (Figure 37–6). Once the mRNA binds to the ribosome, the ribo- tion complex and blocking protein synthesis. some must locate the initiation codon thereby setting the correct reading frame on the mRNA, and translation begins. This pro- Formation of the 48S Initiation Complex cess involves tRNA, rRNA, mRNA, and at least 10 eukaryotic As described in Chapter 36, the 5′ termini of mRNA mole- initiation factors (eIFs), some of which have multiple (three to cules in eukaryotic cells are “capped.” The 7meG-cap facilitates eight) subunits. Also involved are GTP, ATP, and amino acids. the binding of mRNA to the 43S preinitiation complex. A cap- Initiation can be divided into three steps, all of which are obliga- binding protein complex, eIF-4F (4F), which consists of torily preceded by dissociation of the 80S ribosome into its con- eIF-4E (4E) and the eIF-4G (4G)-eIF-4A (4A) complex, binds stituent 40S and 80S subunits: (1) binding of a ternary complex to the cap through the 4E protein. Subsequently eIF-4B (4B) consisting of the initiator methionyl-tRNA (met-tRNAi), binds and reduces the complex secondary structure of the 5′ GTP, and eIF-2 to the 40S ribosome to form the 43S preini- end of the mRNA through its ATP-dependent helicase activity. tiation complex; (2) binding of mRNA to the 40S preinitiation The association of mRNA with the 43S preinitiation complex complex to form the 48S initiation complex; and (3) combina- to form the 48S initiation complex requires ATP hydrolysis. tion of the 48S initiation complex with the 60S ribosomal sub- eIF-3 is a key protein because it binds with high affinity to the unit to form the 80S initiation complex. 4G component of 4F, and links this complex to the 40S ribo- somal subunit. Following association of the 43S preinitiation Ribosomal Dissociation complex with the mRNA cap, and reduction (“melting”) of the Prior to initiation, 80S ribosomes dissociate into component secondary structure near the 5′ end of the mRNA through the 40S and 60S subunits during translation termination (see fol- action of the 4B helicase and ATP, the complex translocates lowing discussion). Dissociation allows these components to 5′ → 3′ and scans the mRNA for a suitable initiation codon. participate in subsequent rounds of translation. Two initiation Generally, this is the 5′-most AUG, but the precise initiation  STEPS IN THE REFORMATION OF THE 80S INITIATION COMPLEX 1. Dissociation of the ribosome 40S + 60S 80S 2. Ternary complex 1 1A formation 3 Ribosome dissociation 60S Met-tRNAMet i Ternary 40S 3 3. Activation of mRNA complex 1 1A 2 Cap AUG (A)n 5 ATP TP 4 = 4E + 4G 4A 4F Met i P B PAB 2 PAB 43S Preinitiation 3 4F Cap AUG (A)n complex 5 1 2B 1A 2 ATP 4B ADP + Pi Met i PAB 4F Cap AUG (A)n 4B 2B 2 PAB 4F Cap 48S Initiation C A AUG (A)n complex 4B 3 1 P GDP 1A 2 5 Met i GTP P ATP ATP-dependent scanning to locate AUG initiation codon ADP + Pi 2B 2 PAB 4F C ap Cap AUG (A)n 3 4B 1 AUG codon recognition 5 1A 2 Met i 2B 5B 2 + Pi 60S 4B 5 1 3 PAB 4F C Cap AUG (A)n 5B E site e A site 4. Active 80S complex Met i 80S Initiation complex P site Elongation 5B 1A FIGURE 37–6 Diagrammatic representation of the initiation phase of protein synthesis on a eukaryotic mRNA. Eukaryotic mRNAs contain a 5′ 7meG-cap (Cap) and 3′ poly(A) terminal [(A)n] as shown. Translation preinitiation complex formation proceeds in several steps: (1) Dissociation of the 80S complex to component 40S and 60S subunits, a process facilitated by binding of factors eIF1, eIF1A, and eIF3 to the ribosomal 40S subunit (top). (2) Formation of the 43S preinitiation complex, a ternary complex consisting of met-tRNAi and GTP-bound to the initiation factor eIF-2 (eIF-2-GTP; left). This complex is then bound by the eIF5 initiation factor forming the complete 43S preinitiation complex. (3) Activation of 5′-capped mRNA and formation of the 48S initiation complex. mRNA is bound via its 5′-cap by eIF4F (composed of eIF4E, eIF4G, and eIF4A factors) and 3′ Poly(A) tail by Poly A binding (PAB) protein forming the 48S initiation complex. ATP hydrolysis-dependent 5′ to 3′ mRNA scanning enables location of the initiation codon AUG, which is then bound by met-tRNAi. (4) Following addition of GTP-bound eI5B and dissociation of eIF1, eIF2-GDP, eIF3, and eIF5, formation of the 80S initiation complex occurs when a recycled 60S ribosomal subunit joins the 48S complex. This reaction positions the initiator met-tRNAi within the P-Site of the active 80S initiation complex formation induces dissociation of eIF1A and GDP-bound eIF5B (see text for details). This complex is now compe- tent for translation initiation. (GTP, ; GDP, °); the various initiation factors appear in abbreviated form as circles or squares, for example, eIF-3, (➂), eIF-4F, (4F), ( ). 4 F is a complex consisting of 4E and 4A bound to 4G (see Figure 37–7). Note that the “circular” structure of mRNA illustrated in Figure 37–7 is thought to be the actual form of mRNA on which steps 1 to 4 actually occur. 411 412 SECTION VII Structure, Function, & Replication of Informational Macromolecules codon is determined by so-called Kozak consensus sequences protein–protein interactions between general and specific that surround the AUG initiation codon: mRNA translational repressors and eIF-4E result in m7G cap- dependent translation control (Figure 37–8). –3 –2 +1 +4 Formation of the 80S Initiation Complex The binding of the 60S ribosomal subunit to the 48S initiation GCCPu A C C AUG G complex involves hydrolysis of the GTP bound to eIF-2 by eIF-5. This reaction results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the Role of the Poly(A) Tail in Initiation 80S ribosome. At this point, the met-tRNAi is on the P site of the Biochemical and genetic experiments have revealed that the ribosome, ready for the elongation cycle to commence. 3′ poly(A) tail and the poly(A) binding protein, PAB, are both required for efficient initiation of protein synthesis. Fur- ther studies showed that the poly(A) tail stimulates recruit- The Regulation of eIF-4E Controls the ment of the 40S ribosomal subunit to the mRNA through a Rate of Initiation complex set of interactions. PAB (Figure 37–7) bound to the The 4F complex is particularly important in controlling the rate poly(A) tail, interacts with eIF-4G, and 4E subunits of cap- of protein translation. As described earlier, 4F is a complex con- bound eIF-4F to form a circular structure that helps direct the sisting of 4E, which binds to the m7G cap structure at the 5′ end 40S ribosomal subunit to the 5′ end of the mRNA and also of the mRNA, and 4G, which serves as a scaffolding protein. In likely stabilizes mRNAs from exonucleolytic degradation. This addition to binding 4E, 4G binds to eIF-3, which links the complex helps explain how the cap and poly(A) tail structures have a to the 40S ribosomal subunit. It also binds 4A and 4B, the ATPase- synergistic effect on protein synthesis. Indeed, differential helicase complex that helps unwind the RNA (see Figure 37–8). Released newly 60S synthesized + polypeptide chain + 40S 80S 4A 5 XppG 7me 4E 4G GUA AA U PAB PAB PAB PAB 3 HO-AAAAAAA(A) A n Nascent polypeptide chain FIGURE 37–7 Schematic illustrating the circularization of mRNA through protein–protein interactions between 7meG cap-bound elF4F and poly(A) tail-bound poly(A) binding protein. elF4F, composed of elF4A, 4E, and 4G subunits binds the mRNA 5′-7meG “Cap” (7meGpppX-) upstream of the translation initiation codon (AUG) with high affinity. The elF4G subunit of the complex also binds poly(A) binding protein (PAB) with high affinity. Since PAB is bound tightly to the mRNA 3′-poly(A) tail (5′-(X)nA(A)n AAAAAAAOH 3′), circularization results. Shown are multiple 80S ribosomes that are in the process of translating the circularized mRNA into protein (black curlicues), forming a polysome. On encountering a ter- mination codon (here UAA), translation termination occurs leading to release of the newly translated protein and dissociation of the 80S ribosome into 60S, 40S subunits. Dissociated ribosomal subunits can recycle through another round of translation (see Figures 37–6 and 37–10).  CHAPTER 37 Protein Synthesis & the Genetic Code 413 PO4 results in its dissociation from 4E, and it cannot rebind until critical sites are dephosphorylated. These effects on the activa- 4E-BP tion of 4E explain in part how insulin causes a marked post- 4E-BP transcriptional increase of protein synthesis in liver, adipose, 4E 4E PO4 and muscle tissue. Insulin (kinase activation) 4G Elongation Is Also a Multistep, 4A Accessory Factor-Facilitated Process Elongation is a cyclic process on the ribosome in which one amino acid at a time is added to the nascent peptide chain (Figure 37–9). The peptide sequence is determined by the PO4 order of the codons in the mRNA. Elongation involves several 4A Active eIF-4F steps catalyzed by proteins called elongation factors (EFs). 4E 4G = 4F complex These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, (3) translocation of the ribosome on the mRNA, and (4) expulsion of the deacylated tRNA from the P- and E-sites. PAB 4F Cap AUG (A)n Binding of Aminoacyl-tRNA to the A Site Active translation In the complete 80S ribosome formed during the process of ini- tiation, both the A site (aminoacyl or acceptor site) and E site FIGURE 37–8 Activation of eIF-4E by insulin and formation (deacylated tRNA exit site) are free (see Figure 37–6). The bind- of the cap-binding eIF-4F complex. The 4F-cap mRNA complex is depicted as in Figures 37–6 and 37–7. The 4F complex consists of ing of the appropriate aminoacyl-tRNA in the A site requires eIF-4E (4E), eIF-4A (4A), and eIF-4G (4G). 4E is inactive when bound proper codon recognition. Elongation factor 1A (EF1A) forms by one of a family of binding proteins (4E-BPs). Insulin and mitogenic a ternary complex with GTP and the entering aminoacyl-tRNA growth polypeptides, or growth factors (eg, IGF-1, PDGF, interleu- (see Figure 37–9). This complex then allows the correct aminoacyl- kin-2, and angiotensin II) activate the PI3 kinase/AKT kinase signal- tRNA to enter the A site with the release of EF1A-GDP and ing pathways, which activate the mTOR kinase; this results in the phosphorylation of 4E-BP (see Figure 42–8). Phosphorylated 4E-BP phosphate. GTP hydrolysis is catalyzed by an active site on dissociates from 4E, and the latter is then able to form the 4F complex the ribosome; hydrolysis induces a conformational change in and bind to the mRNA cap. These growth polypeptides also induce the ribosome concomitantly increasing affinity for the tRNA. phosphorylation of 4G itself by the mTOR and MAP kinase pathways As shown in Figure 37–9, EF1A-GDP then recycles to EF1A- (see Chapter 42). Phosphorylated 4F binds much more avidly to the GTP with the aid of other soluble protein factors and GTP. cap than does nonphosphorylated 4F, which stimulates 48S initiation complex formation and hence translation. Peptide Bond Formation 4E is responsible for recognition of the mRNA cap struc- The α-amino group of the new aminoacyl-tRNA in the A site ture, a rate-limiting step in translation. This process is further carries out a nucleophilic attack on the esterified carboxyl regulated by phosphorylation (see Figure 37–8). Insulin and group of the peptidyl-tRNA occupying the P site (peptidyl mitogenic growth factors result in the phosphorylation of 4E or polypeptide site). At initiation, this site is occupied by the on Ser209 (or Thr210). Phosphorylated 4E binds to the cap initiator met-tRNAi. This reaction is catalyzed by a peptidyl much more avidly than does the nonphosphorylated form, transferase, a component of the 28S RNA of the 60S ribo- thus enhancing the rate of initiation. Components of the MAP somal subunit. This is another example of ribozyme activity kinase, PI3K, mTOR, RAS, and S6 kinase signaling pathways and indicates an important—and previously unsuspected— (see Figure 42–8) can all, under appropriate conditions, be direct role for RNA in protein synthesis (Table 37–3). Because involved in these regulatory phosphorylation reactions. the amino acid on the aminoacyl-tRNA is already “activated,” The activity of 4E is modulated in a second way, and this no further energy source is required for this reaction. The also involves phosphorylation; a set of proteins bind to and inac- reaction results in attachment of the growing peptide chain to tivate 4E. These proteins include 4E-BP1 (BP1, also known as the tRNA in the A site. PHAS-1) and the closely related proteins 4E-BP2 and 4E-BP3. BP1 binds with high affinity to 4E. The 4E-BP1 association pre- vents 4E from binding to 4G (to form 4F). Since this interaction Translocation is essential for the binding of 4F to the ribosomal 40S subunit The now deacylated tRNA is attached by its anticodon to the and for correctly positioning it on the capped mRNA, BP-1 P site at one end and by its open 3′ CCA tail to the E site on effectively inhibits translation initiation. the large ribosomal subunit (middle portion of Figure 37–9). Insulin and other growth factors result in the phosphory- At this point, elongation factor 2 (EF2) binds to and displaces lation of BP-1 at seven unique sites. Phosphorylation of BP-1 the peptidyl tRNA from the A site to the P site. In turn, the 414 SECTION VII Structure, Function, & Replication of Informational Macromolecules TABLE 37–3 Evidence That rRNA Is a Peptidyl Transferase Ribosomes can make peptide bonds (albeit inefficiently) even when proteins are removed or inactivated. Certain parts of the rRNA sequence are highly conserved in all species. These conserved regions are on the surface of the RNA molecule. RNA can be catalytic in many other chemical reactions. Mutations that result in antibiotic resistance at the level of protein synthesis are more often found in rRNA than in the protein compo- nents of the ribosome. X-ray crystal structure of large subunit bound to tRNAs suggests detailed mechanism. deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leav- ing the A site open for occupancy by another ternary complex of amino acid tRNA–EF1A-GTP and another cycle of elongation. The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed peptidyl-tRNA in the A site into the P site by EF2 simi- larly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second; prokary- otic ribosomes incorporate as many as 18 per second. Thus, the energy requiring process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. Termination Occurs When a Stop Codon Is Recognized In comparison to initiation and elongation, termination is a relatively simple process (Figure 37–10). After multiple cycles of elongation culminating in polymerization of the specific amino acids into a protein molecule, the stop or terminating codon of mRNA (UAA, UAG, UGA) appears in the A site. Nor- mally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factor 1 (RF1) recognizes that a stop codon resides in the A site (see Figure 37–10). RF1 is bound by a complex consisting of releasing factor 3 (RF3) with bound GTP. This complex, with the peptidyl transferase, promotes hydrolysis of the bond between the peptide and the tRNA occupying the P site. Thus, a water molecule rather than an amino acid is added. This hydrolysis releases the protein FIGURE 37–9 Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labeled and the tRNA from the P site. On hydrolysis and release, the n − 1, n, n + 1, etc., represent the amino acid residues of the newly 80S ribosome dissociates into its 40S and 60S subunits, which formed protein molecule (in N-terminal to C-terminal orientation) are then recycled (see Figure 37–7). Therefore, the releasing and the corresponding codons in the mRNA. EFIA and EF2 represent factors are proteins that hydrolyze the peptidyl-tRNA bond elongation factors 1 and 2, respectively. The peptidyl-tRNA, amino- when a stop codon occupies the A site. The mRNA is then acyl-tRNA, and exit sites on the ribosome are represented by P site, A site, and E site, respectively. released from the ribosome, which dissociates into its compo- nent 40S and 60S subunits, and another cycle can be repeated (see Figure 37–6).  CHAPTER 37 Protein Synthesis & the Genetic Code 415 STOP 5-Cap 3 (A)n E C A + Releasing factor (RF1) site P site site + GTP Releasing factor (RF3) N STOP 5-Cap 3 (A)n GTP H2O E C A site P site site N 5-CAP 3 (A)n N C + + 40S + 60S + + GDP + Pi Peptide RF1 RF3 tRNA FIGURE 37–10 Diagrammatic representation of the termination process of protein synthesis. The 60S ribosomal peptidyl-tRNA, aminoacyl-tRNA, and exit sites are indicated as P site, A site, and E site, respectively. The termination (stop) codon is indicated by the three verti- cal bars and STOP. Releasing factor RF1 binds to the stop codon in the A site. Releasing factor RF3, with bound GTP, binds to RF1. Hydrolysis of the peptidyl-tRNA complex is shown by the entry of water (H2O); arrow. N and C indicate the amino- and carboxy-terminal amino acids of the nascent polypeptide chain, respectively, and illustrate the polarity of protein synthesis. Termination results in release of the mRNA, the newly synthesized protein (N- and C-termini; N, C), free tRNA, 40S and 60S subunits, as well as RF1, GDP-bound RF3, and inorganic Pi, as shown at bottom. Polysomes Are Assemblies of form a polyribosome, or “polysome” (see Figure 37–7). In an unrestricted system, the number of ribosomes attached to an Ribosomes mRNA (and thus the size of polyribosomes) correlates posi- Many ribosomes can translate the same mRNA molecule simul- tively with the length of the mRNA molecule. taneously. Because of their relatively large size, the ribosome Polyribosomes actively synthesizing proteins can exist as particles cannot attach to an mRNA any closer than 35 nucleo- free particles in the cellular cytoplasm or may be attached to tides apart. Multiple ribosomes on the same mRNA molecule sheets of membranous cytoplasmic structures referred to as 416 SECTION VII Structure, Function, & Replication of Informational Macromolecules the endoplasmic reticulum (ER). Attachment of the particulate within P bodies. These proteins range from mRNA binding polyribosomes to the ER is responsible for its “rough” appear- proteins to mRNA decapping enzymes, RNA helicases, and ance as seen by electron microscopy. The proteins synthesized by RNA exonucleases (5′-3′ and 3′-5′), to components involved the attached polyribosomes are extruded into the cisternal space in miRNA function and mRNA quality control. Incorporation between the sheets of rough ER and are exported from there. of an mRNA into such complexes is not an unequivocal “death Some of the protein products of the rough ER are packaged by sentence.” Indeed, though the mechanisms are not yet fully the Golgi apparatus for eventual export (see Figures 49–2, 49–6). understood, certain mRNAs appear to be temporarily stored in The polyribosomal particles free in the cytosol are responsible P bodies (or SGs) and then retrieved and utilized for protein for the synthesis of proteins required for intracellular functions. translation. This molecular behavior suggests that the cytoplas- mic functions of mRNA (translation and degradation) are con- Nontranslating mRNAs Can Form trolled, at least in part, by the dynamic interaction of mRNA with polysomes and P body/SG protein/enzyme constituents. Ribonucleoprotein Particles That Accumulate in Cytoplasmic Organelles The Machinery of Protein Synthesis Can Termed P Bodies or Stress Granules Respond to Environmental Threats mRNAs, bound by specific packaging proteins and exported Ferritin, an iron-binding protein, prevents ionized iron (Fe2+) from the nucleus as ribonucleoproteins particles (mRNPs) from reaching toxic levels within cells. Elemental iron stimu- sometimes do not immediately associate with ribosomes to lates ferritin synthesis by causing the release of a cytoplasmic be translated. Alternatively, under certain conditions where protein that binds to a specific region in the 5′ nontranslated translation is slowed or stopped (cellular stress or developmen- region of ferritin mRNA. Disruption of this protein-mRNA tal cues among other signals/conditions) select, untranslated interaction activates ferritin mRNA and results in its translation. mRNAs can associate with a range of specific RNAs and pro- This mechanism provides for rapid control of the synthesis of teins to form P bodies and the related stress granules (SG). a protein that sequesters Fe2+, a potentially toxic molecule (see These structures are biomolecular condensates composed of Figures 52–7 and 52–8). Similarly, environmental stress and interacting RNAs and proteins. P bodies can readily be visual- starvation inhibit the positive roles of mTOR (see Figures 37–8 ized via immunohistochemistry using appropriate antibodies and 42–8) on promoting activation of eIF-4F and 48S com- (Figure 37–11). These cytoplasmic structures are related to plex formation. similar small mRNA-containing granules found in neurons and certain maternal cells. Overall P bodies (and SGs) are thought to Many Viruses Co-opt the Host Cell contribute importantly to mRNA metabolism. Over 35 distinct Protein Synthesis Machinery proteins have been suggested to reside exclusively or extensively The protein synthesis machinery can also be modified in del- eterious ways. Viruses replicate by using host cell processes, including those involved in protein synthesis. Some viral P bodies mRNAs are translated much more efficiently than those of the host cell (eg, encephalomyocarditis virus). Others, such as reo- virus and vesicular stomatitis virus, replicate efficiently, and thus their very abundant mRNAs have a competitive advan- tage over host cell mRNAs for limited translation factors. Other viruses inhibit host cell protein synthesis by preventing the association of mRNA with the 40S ribosome. Poliovirus and other picornaviruses gain a selective advan- tage by disrupting the function of the 4F complex. The mRNAs of these viruses do not have a cap structure to direct the bind- ing of the 40S ribosomal subunit (see earlier). Instead, the 40S ribosomal subunit contacts an internal ribosomal entry site FIGURE 37–11 The P body is a cytoplasmic structure (IRES) in a reaction that requires 4G but not 4E. The virus gains involved in mRNA metabolism. Shown is a photomicrograph of two a selective advantage by having a protease that attacks 4G and mammalian cells in which a single distinct protein constituent of the P body has been visualized using the cognate-specific fluorescently removes the amino terminal 4E binding site. Now the 4E-4G labeled antibody. P bodies appear as small red circles of varying size complex (4F) cannot form, so the 40S ribosomal subunit can- throughout the cytoplasm. The cell plasma membranes are indicated not be directed to host capped mRNAs, abolishing host cell pro- by a solid white line, nuclei by a dashed line. Nuclei were counter- tein synthesis. The 4G fragment can direct binding of the 40S stained using a fluorescent dye with different fluorescence excitation/ ribosomal subunit to IRES-containing mRNAs, so viral mRNA emission spectra from the labeled antibody used to identify P bodies; the nuclear stain intercalates between the DNA base pairs and appears translation is very efficient (Figure 37–12). These viruses also as blue/green. Modified from http://www.mcb.arizona.edu/parker/ promote the dephosphorylation of BP1 (PHAS-1), thereby WHAT/what.htm. (Reproduced with permission from Dr. Roy Parker.) decreasing cap (4E)-dependent translation (see Figure 37–8).  CHAPTER 37 Protein Synthesis & the Genetic Code 417 4G 4E Cap AUG (Cellular) Partial Proteolysis of eIF4 4G 4E 4G IRES AUG (Viral) 4E Polio virus protease Cap AUG (Cellular) 4G 4G 4E IRES AUG (Viral) FIGURE 37–12 Picornaviruses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). However, 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of certain viral mRNAs. To gain selective advantage, some viruses (eg, poliovirus) express a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap (ie, host cell mRNAs). The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example. Other viruses utilize distinct processes to effect selective initiation of translation on their cognate viral mRNAs via IRES elements. POSTTRANSLATIONAL Specific enzymes then carry out hydroxylations and oxida- tions of specific amino acid residues within the procollagen PROCESSING AFFECTS THE molecules to provide cross-links for greater stability. Amino ACTIVITY OF MANY PROTEINS terminal peptides are cleaved off the molecule to form the final Some animal viruses, notably HIV, poliovirus, and hepatitis product—a strong, insoluble collagen molecule. Many other virus, synthesize long polycistronic proteins from one long posttranslational modifications of proteins occur. Covalent mRNA molecule. The viral protein molecules translated from modification by acetylation, phosphorylation, methylation, these long mRNAs are subsequently cleaved at defined sites ubiquitylation, and glycosylation is common (see Chapter 5; to provide the several specific viral proteins required for viral Table 35–1). function. In animal cells, many cellular proteins are synthesized from the mRNA template as a precursor molecule, which then must be modified to achieve the active protein. The prototype MANY ANTIBIOTICS WORK is insulin, a small protein having two polypeptide chains with BY SELECTIVELY INHIBITING interchain and intrachain disulfide bridges. The molecule is synthesized as a single chain precursor, or prohormone, which PROTEIN SYNTHESIS IN BACTERIA folds to allow the formation of specific, intra- or intermolecular Ribosomes in bacteria and in the mitochondria of higher disulfide bridges (see intramolecular disulfide bonds in insulin; eukaryotic cells differ from the mammalian ribosome Figure 41–12). A specific protease then clips out the segment described in Chapter 34. The bacterial ribosome is smaller that connects the two chains which form the functional insulin (70S vs 80S) and has a different, somewhat simpler comple- molecule (see Figure 41–12). ment of RNA and protein molecules. This difference can be Many other peptides are synthesized as precursor pro- exploited for clinical purposes because many effective anti- proteins that require modifications before attaining biologic biotics interact specifically with the proteins and RNAs of activity. Many of the posttranslational modifications involve prokaryotic ribosomes and thus only inhibit bacterial pro- the removal of amino terminal amino acid residues by specific tein synthesis. This results in growth arrest (ie, bacteriostatic aminopeptidases (see Figure 41–14). By contrast, collagen, an action) or death (ie, bactericidal action) of the bacterium. The abundant protein in the extracellular spaces of higher eukary- most useful members of this class of antibiotics (eg, tetracy- otes, is synthesized as procollagen. Three procollagen poly- clines, lincomycin, erythromycin, and chloramphenicol) peptide molecules, frequently not identical in sequence, align do not interact with components of eukaryotic ribosomes themselves in a particular way that is dependent on the exis- and thus are not toxic to eukaryotes. Tetracycline prevents tence of specific amino terminal peptides (see Figure 5–11). the binding of aminoacyl-tRNAs to the bacterial ribosome A 418 SECTION VII Structure, Function, & Replication of Informational Macromolecules site. Chloramphenicol works by binding to 23S rRNA, which cell membrane rather than to insensitivity of mouse EF-2 to is interesting in view of the newly appreciated role of rRNA in diphtheria toxin-catalyzed ADP-ribosylation by NAD. peptide bond formation through its peptidyl transferase activity. Ricin, an extremely toxic molecule isolated from the castor It should be mentioned that the close similarity between pro- bean, inactivates eukaryotic 28S ribosomal RNA by catalyzing karyotic and mitochondrial ribosomes can lead to complica- the N-glycolytic cleavage or removal of a single adenine. tions in the use of some antibiotics. Many of these compounds—puromycin and cycloheximide Other antibiotics inhibit protein synthesis on all ribo- in particular—are not clinically useful but have been important somes (puromycin) or only on those of eukaryotic cells in the laboratory as a tool to elucidate the role of protein synthe- (cycloheximide). Puromycin (Figure 37–13) is a structural sis in the regulation of metabolic processes, particularly enzyme analog of tyrosinyl-tRNA. Puromycin is incorporated via the induction by hormones. A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits pro- SUMMARY The flow of genetic information generally follows the sequence tein synthesis in both prokaryotes and eukaryotes. Cyclohexi- DNA → RNA → protein. mide inhibits peptidyl transferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. Ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA) are directly involved in protein synthesis. Diphtheria toxin, an exotoxin of Corynebacterium diphtheriae infected with a specific lysogenic phage, catalyzes The information in mRNA is a continuous linear array of codons, the ADP-ribosylation of EF-2 on the unique amino acid diph- each of which is three nucleotides long. thamide (a posttranslationally modified version of histidine) The mRNA is read continuously from a start (AUG) to in mammalian cells. This modification inactivates EF-2 and termination (UAA, UAG, UGA) codon. thereby specifically inhibits mammalian protein synthesis. The open reading frame, or ORF, of the mRNA is the series of Many animals (eg, mice) are resistant to diphtheria toxin. This contiguous codons (AUG to STOP), each codon specifying a resistance is due to inability of diphtheria toxin to cross the certain amino acid, which determines the precise amino acid sequence of the protein. Protein synthesis, like DNA and RNA synthesis, follows the 5′ N(CH3)2 to 3′ polarity of mRNA and can be divided into three processes: initiation, elongation, and termination. N N Mutant proteins arise when base substitutions result in codons that specify a different amino acid at a given position, when N a stop codon results in a truncated protein, or when base HOCH2 O N additions or deletions alter the reading frame, so different codons are read. H H H H A variety of compounds, including several antibiotics, inhibit protein synthesis by affecting one or more of the steps involved NH OH in protein synthesis. O C CH CH2 OCH3 NH2