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25-31). The twogenes overlap, with pol encoded by the readingframe in which each codon is offset to the left byone base pair (-1 reading frame) relative to gag(Fig. 1). The product of the pol gene (reverse transcriptase; p. 882) is translated initially as a largergag-pol fusion protein using the same mRNA usedfor the gag protein alone. This fusion protein islater trimmed to the mature reverse transcriptaseby proteolytic digestion. The large fusion protein isproduced by a translational frameshift that occursin the overlap region and allows the ribosome tobypass the UAG termination codon at the end ofthe gag gene (shown in red in Fig.
1). This frameshift occurs in about 5% of the translation events,so that the gag-pol fusion protein, and ultimatelyreverse transcriptase, is synthesized at the appropriate level for efficient replication of the viral genome—about 20-fold less than the gag protein. Asimilar mechanism is used to produce both the rLeu — Gly — Leu — Arg — Leu — Thr — Asn — LeuStopU A||G G G|[C U CllC G~Ci[U U G[|A C Al|A A U||U U~A|gag reading frame 5'pol reading frameand y subunits of E.
coli DNA polymerase III fromdnaX gene transcripts (see Table 24-2).An example of the use of this mechanism forregulation occurs in the gene for E. coli release factor 2 (RF2), a protein required for termination ofprotein synthesis at the termination codons UAAand UGA (described later in this chapter).
The26th codon of the gene for RF 2 is UGA, whichwould normally halt protein synthesis. The remainder of the gene is in the +1 reading frame (offset one base pair to the right) relative to this UGAcodon. Low levels of RF 2 lead to a translationalpause at this codon, because UGA is not recognizedas a termination codon unless RF2 binds to it. Theabsence of RF 2 prevents the termination of proteinsynthesis at this UGA and allows time for a frameshift so that UGA plus the C that follows it (UGAC)is read as GAC = Asp. Translation then proceedsin the new reading frame to complete synthesis ofRF2. In this way, RF2 regulates its own synthesisin a feedback loop.An especially unusual frameshifting mechanism occurs through the editing of mRNAs prior totranslation.
The genes in mitochondrial DNA thatencode the cytochrome oxidase subunit II in someprotists do not have open reading frames that correspond precisely to the protein product. Instead,the codons specifying the amino terminus of theprotein are in a different reading frame from thecodons specifying the carboxyl terminus.
The problem is corrected not on the ribosome, but by a posttranscriptional editing process in which four uridines are added to create three new codons andshift the reading frame so that the entire gene canbe translated directly, as shown in Figure 2a; theCG G A G G G C C AU A G G G C U C C G C U U G A C A A A U U UHe Figure 1 The gag—pol overlap region in Rous sarcoma virus.Gly — Arg — AlaChapter 26 Protein MetabolismDNA5'LysEditedmRNAA||a A a||A A C||C T G|G T AA A A||Q—ValAsnGluA A A||G U A||G Av\\u GLeuValFigure 2 RNA editing of the transcript of the cytochrome oxidase subunit II gene from mitochondriaof Trypanosoma brucei.-- 3—-U||A U A||C € -U||G G901u(a)mRNA 5'--- A A A G U A G A U U G U A U A C C U G GU3'U U A U A U C U A A U A U A U G G A U A U ,Guide RNA(b)added uridine residues are shown in red.
Only asmall part of the gene (the region affected by editing) is shown. Neither the function nor mechanismof this editing process is understood. A specialclass of RNA molecules encoded by these mitochondria have been detected that have sequences complementary to the final, edited mRNAs. These appear to act as templates for the editing process andare referred to as guide RNAs (Fig. 2b). Note thatthe base pairing involves a number of G=U basepairs (symbolized by blue dots), which are commonin RNA molecules.A distinct form of RNA editing occurs in thegene for the apolipoprotein B component of lowdensity lipoprotein in vertebrates.
One form of apo-Human liver 5'(apoB-100)lipoprotein B, called apoB-100 (Mr 513,000), is synthesized in the liver. A second form, apoB-48 (Mr250,000), is synthesized in the intestine. Both aresynthesized from an mRNA produced from thegene for apoB-100. A cytosine deaminase enzymefound only in the intestine binds to the mRNA atcodon 2,153 (CAA = Gin) and converts the C to a Uto introduce the termination codon UAA at thisposition.
The apoB-48 produced in the intestinefrom the modified mRNA is simply an abbreviatedform (corresponding to the amino-terminal half) ofapoB-100 (Fig. 3). This reaction permits the synthesis of two different proteins from one gene in atissue-specific manner.[C A A|[C U G[|C A GJ[A C A[|U A U[|A U G[|A U A||C A A\\U U U||GA U||C A G||U A U]-Gin — Leu — Gin — Thr — Tyr -Met — lie -Human intestine - - ~|C A Aj[C U G\|C A G|[A C A[|U A U[[A U G|[A U A(apoB-48)- Gin - Leu - Gin - Thr - Tyr - Met HeResidue number2,1462,1482,1502,152Gin — Phe — Asp -Gin -Tyr3'-U U U l | G A UllC A GllU A U[Stop2,1542,156Figure 3 RNA editing of the transcript of the genefor the apolipoprotein B-100 component of lowdensity lipoprotein.Part IV Information PathwaysThe third bases of the arginine codons (A, U, and C) form rather weakhydrogen bonds with the I residue at the first position of the anticodon.Examination of these and other codon-anticodon pairings led Crick toconclude that the third base of most codons pairs rather loosely withthe corresponding base of its anticodons; to use his picturesque word,the third bases of such codons "wobble." Crick proposed a set of fourrelationships called the wobble hypothesis:1.
The first two bases of a codon in mRNA always form strongWatson-Crick base pairs with the corresponding bases of the anticodon in tRNA and confer most of the coding specificity.2. The first base of some anticodons (reading in the 5'->3' direction;remember that this is paired with the third base of the codon)determines the number of codons read by a given tRNA. When thefirst base of the anticodon is C or A, binding is specific and only onecodon is read by that tRNA. However, when the first base is U or G,binding is less specific and two different codons may be read. Wheninosinate (I) is the first, or wobble, nucleotide of an anticodon,three different codons can be read by that tRNA. This is the maximum number of codons that can be recognized by a tRNA. Theserelationships are summarized in Table 26-5.3.
When an amino acid is specified by several different codons, thosecodons that differ in either of the first two bases require differenttRNAs.4. A minimum of 32 tRNAs are required to translate all 61 codons.Table 26-5 The wobble base of the anticodon determines how manycodons of a given amino acid a tRNA can recognizeIn the following, X and Y denote complementary bases capable of strongWatson-Crick base pairing with each other. The bases in the wobble or 3'position of the codons and 5' position of the anticodons are shaded in red.1.
One codon recognized:Anticodon(3') X-Y~€ (5')(3') X-Y-A (5')Codon2. Two codons recognized:AnticodonCodon3. Three codons recognized:AnticodonCodon(5') Y-X-G (3')(5') Y-X-U (3')(3') X-Y-U (5')(3') X-Y-G (5')(5') Y-X-* (3')(5') Y-X-g (3')(3') X - Y - I (5')(5') Y-X-U (3')cWhat can the reason be for this unexpected complexity of codonanticodon interactions? In brief, the first two bases of a codon confermost of the codon-anticodon specificity. The wobble (or third) base ofChapter 26 Protein Metabolism903the codon contributes to specificity, but because it pairs only looselywith its corresponding base in the anticodon, it permits rapid dissociation of the tRNA from its codon during protein synthesis. If all threebases of mRNA codons engaged in strong Watson-Crick pairing withthe three bases of the tRNA anticodons, tRNAs would dissociate tooslowly and severely limit the rate of protein synthesis.
Codonanticodon interactions optimize both accuracy and speed.Overlapping Genes in Different Reading FramesAre Found in Some Viral DNAsAlthough a given nucleotide sequence can, in principle, be read in anyof its three reading frames, most DNA sequences encode a proteinproduct in only one reading frame. In the coding frame there must beno termination codons, and each codon must correspond to the appropriate amino acid. As illustrated in Figure 26-9, the genetic code imposes strict limits on the numbers of amino acids that can be encodedby the codons of reading frame 2 without changing the amino acidsspecified by reading frame 1.
Sometimes one amino acid (and its corresponding codon) may be substituted for another in reading frame 1 andstill retain the function of the encoded protein, making it more likelythat reading frames 2 or 3 might also encode a useful protein; but eventaking these factors into account, the flexibility in other readingframes is very limited.Figure 26-9 An amino acid sequence specified byone reading frame severely limits the potentialamino acids encoded by any other reading frame,(a) The codons that can exist in reading frame 1 toproduce the indicated amino acid sequence. Most ofthe permitted nucleotide changes (red) are in thethird (wobble) position of each codon. (b) At the topare shown the codons that can exist in readingframe 2 without changing the amino acid sequenceencoded by reading frame 1.
Below are shown thealternative codons that correspond to the alternative mRNA sequences listed in (a). The possibleamino acids that can be encoded by reading frame2 without changing the amino acid sequence encoded by reading frame 1 are in parentheses.Amino acid sequenceReading frame 1Met — His — Phe — Thr — Asn — Arg — Tyr5 ' [A U G | | C A C | | U U U | f A C U | | A AOther mRNA sequencesthat specify the sameamino acid sequenceSerC||C G Cl|U A U||U C C| 3'lC A U[[U U cllA C~c]|A A u][C G UJ[U A c ] [ J J C U[A C A|lC G A[lUC G[A C Gl|C G GllUC A[A G A |1A G|A G Gl|AG CU(a)Reading frame 25' A [U G C[|A C U[[U U A~||C U h\[KAmino acid sequenceOther amino acidsresulting from thealternative mRNAsequences shownabove for readingframe 1Cys — Thr -C C[JG C UJJA U U | C C 3 'Leu — Leu — Thr — Ala — He[A U U l | U C Al[C C AHA U C[|G U UlJA C U[(lie)(Ser)(Pro)(lie)(Val)(Thr)C A A||A C A||G A U||A C A(Thr)(Thr)(Asp)(Gin)C G A||A U A||G G(b)a904Part IV Information PathwaysFigure 26-10 Genes within genes.
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