H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 51
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In all tRNAs, the 3 end ofthe unlooped amino acid acceptor stem has the sequenceCCA, which in most cases is added after synthesis and processing of the tRNA are complete. Several bases in mosttRNAs also are modified after synthesis. Viewed in threeTABLE 4-2 Known Deviations from the Universal Genetic CodeCodonUniversalCodeUnusualCode*OccurrenceUGAStopTrpMycoplasma, Spiroplasma, mitochondria of many speciesCUGLeuThrMitochondria in yeastsUAA, UAGStopGlnAcetabularia, Tetrahymena, Paramecium, etc.UGAStopCysEuplotes*“Unusual code” is used in nuclear genes of the listed organisms and in mitochondrial genes as indicated.SOURCE: S.
Osawa et al., 1992, Microbiol. Rev. 56:229.122CHAPTER 4 • Basic Molecular Genetic MechanismsAmino acid(Phe)H OH2NCCH2CHigh-energyester bondOHH2NOHHOCCOH2NCH212OCH2Net result:Phe is selectedby its codon5AAAUUUmRNA3corresponding tRNA. Step 2 : A three-base sequence in thetRNA (the anticodon) then base-pairs with a codon in the mRNAspecifying the attached amino acid.
If an error occurs in eitherstep, the wrong amino acid may be incorporated into apolypeptide chain. Phe phenylalanine.nucleic acid sequences in mRNA into amino acid sequencesin proteins. Step 1 : An aminoacyl-tRNA synthetase first couplesa specific amino acid, via a high-energy ester bond (yellow), toeither the 2 or 3 hydroxyl of the terminal adenosine in theamino acid. As noted above, however, many cells containfewer than 61 tRNAs.
The explanation for the smaller numberlies in the capability of a single tRNA anticodon to recognizemore than one, but not necessarily every, codon correspondingto a given amino acid. This broader recognition can occur because of nonstandard pairing between bases in the so-calledwobble position: that is, the third (3) base in an mRNA codonand the corresponding first (5) base in its tRNA anticodon.The first and second bases of a codon almost alwaysform standard Watson-Crick base pairs with the third anddimensions, the folded tRNA molecule has an L shape withthe anticodon loop and acceptor stem forming the ends ofthe two arms (Figure 4-22b).Nonstandard Base Pairing Often Occurs BetweenCodons and AnticodonsIf perfect Watson-Crick base pairing were demanded betweencodons and anticodons, cells would have to contain exactly 61different tRNA species, one for each codon that specifies an3(a)Science 147:1462; part (b) from J.
G. Arnez andD. Moras, 1997, Trends Biochem. Sci. 22:211.]CAAAAminoacyl-tRNA▲ FIGURE 4-21 Two-step decoding process for translating(a) Although the exact nucleotide sequencevaries among tRNAs, they all fold into fourbase-paired stems and three loops. The CCAsequence at the 3 end also is found in alltRNAs. Attachment of an amino acid to the 3A yields an aminoacyl-tRNA. Some of the A,C, G, and U residues are modified in mosttRNAs (see key). Dihydrouridine (D) is nearlyalways present in the D loop; likewise,ribothymidine (T) and pseudouridine () arealmost always present in the TCG loop.Yeast alanine tRNA, represented here, alsocontains other modified bases.
The triplet atthe tip of the anticodon loop base-pairs withthe corresponding codon in mRNA. (b) Threedimensional model of the generalized backbone of all tRNAs. Note the L shape of themolecule. [Part (a) see R. W. Holly et al., 1965,CAMP+ PPiAAAAminoacyltRNA synthetase tRNA specific forspecific for PhePhe (tRNAPhe) FIGURE 4-22 Structure of tRNAs.OtRNAPhe bindsto the UUU codonLinkage ofPhe to tRNAPheATPHD = dihydrouridineI = inosineT = ribothymidine = pseudouridinem = methyl groupD loopDG AmGUU G C GCGG C G CG D Am2GAnticodon loopCUCCCUU 1IACC5 AAcceptorGCstemGCGUCGGCTCGloopUUGCU UAA G G C CGU C C G GCT GA CAG DG VariableGGloopG2Gml3CAnticodonC C G3321mRNA5Codon(b)TCG loopAcceptor stemC5CD loopVariableloopAnticodon loopA34.4 • The Three Roles of RNA in Translationsecond bases, respectively, of the corresponding anticodon,but four nonstandard interactions can occur between basesin the wobble position. Particularly important is the G·Ubase pair, which structurally fits almost as well as the standard G·C pair.
Thus, a given anticodon in tRNA with G inthe first (wobble) position can base-pair with the two corresponding codons that have either pyrimidine (C or U) in thethird position (Figure 4-23). For example, the phenylalaninecodons UUU and UUC (5n3) are both recognized by thetRNA that has GAA (5n3) as the anticodon. In fact, anytwo codons of the type NNPyr (N any base; Pyr pyrimidine) encode a single amino acid and are decoded bya single tRNA with G in the first (wobble) position of theanticodon.Although adenine rarely is found in the anticodon wobbleposition, many tRNAs in plants and animals contain inosinetRNA35If these bases are infirst, or wobble, position ofanticodon3 21CAGUI1235 mRNA 3GUCUAGCAU5 mRNA 31233 21then the tRNA mayrecognize codons inmRNA having thesebases in third positionIf these bases are inthird, or wobble, positionof codon of an mRNAC A G UGIUICUAGIthen the codon maybe recognized by atRNA having thesebases in first positionof anticodon53tRNA▲ FIGURE 4-23 Nonstandard codon-anticodon base pairingat the wobble position.
The base in the third (or wobble)position of an mRNA codon often forms a nonstandard basepair with the base in the first (or wobble) position of a tRNAanticodon. Wobble pairing allows a tRNA to recognize more thanone mRNA codon (top); conversely, it allows a codon to berecognized by more than one kind of tRNA (bottom), althougheach tRNA will bear the same amino acid. Note that a tRNAwith I (inosine) in the wobble position can “read” (becomepaired with) three different codons, and a tRNA with G or U inthe wobble position can read two codons. Although A istheoretically possible in the wobble position of the anticodon,it is almost never found in nature.123(I), a deaminated product of adenine, at this position. Inosine can form nonstandard base pairs with A, C, and U.
AtRNA with inosine in the wobble position thus can recognizethe corresponding mRNA codons with A, C, or U in the third(wobble) position (see Figure 4-23). For this reason, inosinecontaining tRNAs are heavily employed in translation of thesynonymous codons that specify a single amino acid. For example, four of the six codons for leucine (CUA, CUC, CUU,and UUA) are all recognized by the same tRNA with the anticodon 3-GAI-5; the inosine in the wobble position formsnonstandard base pairs with the third base in the four codons.In the case of the UUA codon, a nonstandard G·U pair alsoforms between position 3 of the anticodon and position 1 ofthe codon.Aminoacyl-tRNA Synthetases Activate AminoAcids by Covalently Linking Them to tRNAsRecognition of the codon or codons specifying a given aminoacid by a particular tRNA is actually the second step in decoding the genetic message.
The first step, attachment of theappropriate amino acid to a tRNA, is catalyzed by a specificaminoacyl-tRNA synthetase. Each of the 20 different synthetases recognizes one amino acid and all its compatible, orcognate, tRNAs. These coupling enzymes link an amino acidto the free 2 or 3 hydroxyl of the adenosine at the 3 terminus of tRNA molecules by an ATP-requiring reaction. Inthis reaction, the amino acid is linked to the tRNA by a highenergy bond and thus is said to be activated. The energy ofthis bond subsequently drives formation of the peptide bondslinking adjacent amino acids in a growing polypeptide chain.The equilibrium of the aminoacylation reaction is driven further toward activation of the amino acid by hydrolysis of thehigh-energy phosphoanhydride bond in the released pyrophosphate (see Figure 4-21).Because some amino acids are so similar structurally,aminoacyl-tRNA synthetases sometimes make mistakes.These are corrected, however, by the enzymes themselves,which have a proofreading activity that checks the fit in theiramino acid–binding pocket.
If the wrong amino acid becomes attached to a tRNA, the bound synthetase catalyzesremoval of the amino acid from the tRNA. This crucial function helps guarantee that a tRNA delivers the correct aminoacid to the protein-synthesizing machinery. The overall errorrate for translation in E. coli is very low, approximately 1 per50,000 codons, evidence of the importance of proofreadingby aminoacyl-tRNA synthetases.Ribosomes Are Protein-Synthesizing MachinesIf the many components that participate in translatingmRNA had to interact in free solution, the likelihood of simultaneous collisions occurring would be so low that therate of amino acid polymerization would be very slow. Theefficiency of translation is greatly increased by the binding ofthe mRNA and the individual aminoacyl-tRNAs to the most124CHAPTER 4 • Basic Molecular Genetic Mechanismsabundant RNA-protein complex in the cell, the ribosome,which directs elongation of a polypeptide at a rate of three tofive amino acids added per second.
Small proteins of100–200 amino acids are therefore made in a minute or less.On the other hand, it takes 2–3 hours to make the largestknown protein, titin, which is found in muscle and containsabout 30,000 amino acid residues. The cellular machine thataccomplishes this task must be precise and persistent.With the aid of the electron microscope, ribosomes werefirst discovered as small, discrete, RNA-rich particles in cellsthat secrete large amounts of protein. However, their role inprotein synthesis was not recognized until reasonably pureribosome preparations were obtained.
In vitro radiolabelingexperiments with such preparations showed that radioactiveamino acids first were incorporated into growing polypeptide chains that were associated with ribosomes before appearing in finished chains.A ribosome is composed of three (in bacteria) or four (ineukaryotes) different rRNA molecules and as many as 83proteins, organized into a large subunit and a small subunit(Figure 4-24).
The ribosomal subunits and the rRNA molecules are commonly designated in Svedberg units (S), ameasure of the sedimentation rate of suspended particles cen-ProteinsrRNAtrifuged under standard conditions. The small ribosomalsubunit contains a single rRNA molecule, referred to as smallrRNA. The large subunit contains a molecule of large rRNAand one molecule of 5S rRNA, plus an additional moleculeof 5.8S rRNA in vertebrates. The lengths of the rRNA molecules, the quantity of proteins in each subunit, and consequently the sizes of the subunits differ in bacterial andeukaryotic cells.
The assembled ribosome is 70S in bacteriaand 80S in vertebrates. But more interesting than these differences are the great structural and functional similaritiesbetween ribosomes from all species. This consistency is another reflection of the common evolutionary origin of themost basic constituents of living cells.The sequences of the small and large rRNAs from severalthousand organisms are now known. Although the primarynucleotide sequences of these rRNAs vary considerably, thesame parts of each type of rRNA theoretically can form basepaired stem-loops, which would generate a similar threedimensional structure for each rRNA in all organisms.
Theactual three-dimensional structures of bacterial rRNAs fromThermus thermopolis recently have been determined by xray crystallography of the 70S ribosome. The multiple, muchsmaller ribosomal proteins for the most part are associatedSubunitsAssembledribosomes5S23SProkaryotic+23S(2900 rNTs)Total: 315S(120 rNTs)50S+Total: 2116S16S(1500 rNTs)70S30SEukaryotic (vertebrate)5S28S+5.8STotal: 5028S5S28S : 5.8S(4800 rNTs, 160 rNTs) (120 rNTs)5.8S60S+Total: 3318S(1900 rNTs)▲ FIGURE 4-24 The general structure of ribosomes inprokaryotes and eukaryotes. In all cells, each ribosomeconsists of a large and a small subunit.
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