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Inserting or deleting one basepair (shown here in the mRNA transcript) altersthe sequence of triplets in a nonoverlapping code,as shown, and all amino acids coded by the mRNAfollowing the change are affected. Combining insertion and deletion mutations affects some aminoacids but eventually restores the correct amino acidsequence. Adding or subtracting three nucleotides(not shown) leaves the remaining triplets intact,providing evidence that a codon has three, ratherthan four or five, nucleotides.
The triplet codonsshaded in gray are those transcribed from the original gene; codons shaded in blue are new codonsresulting from the insertion or deletion mutations.mRNA5'|G UG A[[UCl|U( )1Insertion[G U AJ|G C CJlU C A | | C G G||A UDeletion\G U AHC ~C~U||A C ~G]lG A U[.Insertion anddeletionT „Y|G U A|[A G C J l C ' A C[|G G A[[U'>Reading framerestored3'Chapter 26 Protein MetabolismNonoverlappingcode,A U A,,C G A, ,G U213OverlappingcodeA U A C G A G U Cc,iThe amino acid sequence of a protein is therefore defined by a linearsequence of contiguous triplet codons.
The first codon in the sequenceestablishes a reading frame, in which a new codon begins every threenucleotide residues. In this scheme there are three possible readingframes for any given DNA sequence, and each will generally give adifferent sequence of codons (Fig. 26-5). Although it seemed clear thatonly one reading frame was likely to contain the information requiredfor a given protein, the ultimate questions still loomed: What are thespecific three-letter code words for the different amino acids? Howcould they be identified experimentally?Reading frame 15'895Figure 26—4 Overlapping versus nonoverlappingcodes. In nonoverlapping codes, codons do not sharenucleotides.
In the example shown, the consecutivecodons are numbered. In an overlapping code, somenucleotides in the mRNA are shared by differentcodons. A triplet code with maximum overlap, withconsecutive codons defined by the numbered brackets, will have many nucleotides (such as the thirdnucleotide here) shared by three different codons.Note that in an overlapping code, the sequence ofthe first codon limits the possible sequences for thesecond codon. A nonoverlapping code provides muchmore flexibility in the sequence of neighboring codons and ultimately in the possible amino acid sequences designated by the code.
The code used inall living systems is nonoverlapping.[U U Cl|tJ~C G[]G A C||C U G\\G A G|[A U U][C A~~C]JA G U|Reading frame 2- - - t i l | U C U]\C G G|[A C CllU G G|[A G A[]U U C1 \ATC~A\Reading frame 3- - - U U||C U C||G G AJfC C U|[G G A\\G A~UilU C A[|C AIn 1961 Marshall Nirenberg and Heinrich Matthaei reported anobservation that provided the first breakthrough. They incubated thesynthetic polyribonucleotide polyuridylate (designated poly(U)) withan E.
coli extract, GTP, and a mixture of the 20 amino acids in 20different tubes. In each tube a different amino acid was radioactivelylabeled. Poly(U) can be regarded as an artificial mRNA containingmany successive UUU triplets, and it should promote the synthesis ofa polypeptide from only one of the 20 different amino acids—thatcoded by the triplet UUU.
A radioactive polypeptide was formed inonly one of the 20 tubes, that containing radioactive phenylalanine.Nirenberg and Matthaei therefore concluded that the triplet UUUcodes for phenylalanine. The same approach revealed that the synthetic polyribonucleotide polycytidylate or poly(C) codes for formationof a polypeptide containing only proline (polyproline), andpolyadenylate or poly(A) codes for polylysine. Thus the triplet CCCmust code for proline and the triplet AAA for lysine.The synthetic polynucleotides used in such experiments weremade by the action of polynucleotide phosphorylase (p. 880), whichcatalyzes the formation of RNA polymers starting from ADP, UDP,CDP, and GDP. This enzyme requires no template and makes polymerswith a base composition that directly reflects the relative concentrations of the nucleoside 5'-diphosphate precursors in the medium.
Ifpolynucleotide phosphorylase is presented with UDP, it makes onlypoly(U). If it is presented with a mixture of five parts of ADP and one of3'[G~tT - - -Figure 26-5 In a triplet, nonoverlapping code, allmRNAs have three potential reading frames,shaded here in different colors. Note that the triplets, and hence the amino acids specified, are verydifferent in each reading frame.Marshall Nirenberg896Part IV Information PathwaysCDP, it will make a polymer in which about five-sixths of the residuesare adenylate and one-sixth cytidylate. Such a random polymer islikely to have many triplets of the sequence AAA, lesser numbers ofAAC, ACA, and CAA triplets, relatively few ACC, CCA, and CAC triplets, and very few CCC triplets (Table 26-1).
With the use of differentartificial mRNAs made by polynucleotide phosphorylase from differentstarting mixtures of ADP, GDP, UDP, and CDP, the base compositionsof the triplets coding for almost all the amino acids were soon identified. However, these experiments could not reveal the sequence of thebases in each coding triplet.Table 26-1 Incorporation of amino acids into polypeptides inresponse to random polymers of RNA*Amino acidAsparagineGlutamineHistidineLysineProlineThreonineH. Gobind KhoranaObservedfrequency ofincorporation(Lys = 100)Tentative assignmentfor nucleotidecompositiont ofcorresponding codonExpected frequencyof incorporationbased onassignment(Lys - 100)24(A) 2 C2024(A) 2 C206A(C) 2100726(A) 3A(C) 2 , (C) 3(A) 2 C, A(C) 241004.824* Presented here is a summary of data from one of the early experiments designed to elucidatethe genetic code. An RNA synthesized enzymatically, and containing only A and C residues in a5:1 ratio, was used to direct polypeptide synthesis.
Both the identity and quantity of amino acidsincorporated were determined. Based upon the relative abundance of A and C residues in thesynthetic RNA, and if the codon AAA (the most likely) is assigned a frequency of 100, thereshould be three different codons of composition (A)2C, each at a relative frequency of 20; threecodons of composition A(C)2, each at a relative frequency of 4.0; and the codon CCC should occurat a relative frequency of 0.8.
The CCC assignment here was based on information derived fromprior studies with poly(C). Where two tentative codon assignments are made, both are proposedto code for the same amino acid.t Note that these designations of nucleotide composition contain no information on nucleotidesequence.In 1964 Nirenberg and Philip Leder achieved another breakthrough. They found that isolated E. coli ribosomes will bind a specificaminoacyl-tRNA if the corresponding synthetic polynucleotide messenger is present. For example, ribosomes incubated with poly(U) andphenylalanyl-tRNAphe (or Phe-tRNAPhe) will bind both polymers, but ifthe ribosomes are incubated with poly(U) and some other aminoacyltRNA, the aminoacyl-tRNA will not be bound because it will not recognize the UUU triplets in poly(U) (Table 26-2). (Note that by convention, the identity of a tRNA is indicated by a superscript and anaminoacylated tRNA is indicated by a hyphenated name. For example,correctly aminoacylated tRNA*1* is alanyl-tRNA^ 3 or Ala-tRNA^3.
Ifthe tRNA is incorrectly aminoacylated, e.g., with valine, one wouldhave Val-tRNA^.) The shortest polynucleotide that could promotespecific binding of Phe-tRNAPhe was the trinucleotide UUU. By use ofsimple trinucleotides of known sequence it was possible to determinewhich aminoacyl-tRNA bound to each of about 50 of the 64 possibletriplet codons. For some codons, either no aminoacyl-tRNAs wouldbind, or more than one were bound. Another method was needed tocomplete and confirm the entire genetic code.Chapter 26 Protein Metabolism897Table 26-2 Experiment showing that trinucleotides are sufficient toinduce specific binding of aminoacyl-tENAs to ribosomes14Phe-tRNA PheTrinucleotideC-Labeled aminoacyl-tRNAbound to ribosome*Pro-tRNA ProLys-tRNALysUUU4.6AAA07.7CCC000003.1Source Modified from Nirenberg, M & Leder, P (1964) RNA code words and protein synthesis.Science 145, 1399xThe numbers represent factors by which the amount of bound 14C increased when the indicatedtrinucleotide was present, relative to controls in which no trinucleotide was added.At about this time, a complementary approach was provided byH.
Gobind Khorana, who developed methods to synthesize polyribonucleotides with defined, repeating sequences of two to four bases. Thepolypeptides produced using these RNAs as messengers had one or afew amino acids in repeating patterns. These patterns, when combinedwith information from the random polymers used by Nirenberg andcolleagues, permitted unambiguous codon assignments. The copolymer(AC)n, for example, has alternating ACA and CAC codons, regardlessof the reading frame:,A C A , , C A C , , A C A , , C A C , , A C A ,The polypeptide synthesized in response to this polymer was found tohave equal amounts of threonine and histidine.
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