J. Mol. Biol. (1962) 4, 121-122

Chain Growth of Proteins: Some Consequences for theCoding Problem

Previous formulations of the coding problem have contained the implicit assumptionthat amino acids might attach independently to their appropriate loci anywherealong the template, and become fixed there, prior to the formation of peptide bonds.This concept implied that each locus must be demarcated clearly from its neighbor, toprevent misreadings of the code. It was argued that all overlapping segments ofadjacent coding units must be nonsense, i.e. must not correspond to any of thetwenty words in the code. Such considerations led to formulation of non-degeneratecodes like the "comma-less" one of Crick, Griffith & Orgel (1957).

Experiments with specific proteins of mammalian (Bishop, Leahy & Schweet, 1960;Dintzis, 1961) and avian (Steinberg, Vaughan & Anfinsen, 1956) systems, and recentwork in this laboratory with the total proteins of E. coli (Goldstein, 1961) indicatethat protein is assembled, residue by residue, from the N-terminal end. Theseexperiments suggest that at any instant only a single peptide bond can be formed,that between the terminal -COOH of the growing chain and the -NH2 of a correctlypositioned amino acid in the adjacent locus. If this is the case, then the problemof transient interactions of amino acids with any region of the template removedfrom the point of chain growth may be irrelevant; the stepwise mode of assemblywould, by its very nature, preclude a misreading of the code. Consequently, theprincipal restrictions of the comma-less code would be superfluous. Potential codingunits need not be ruled out merely because their overlapping segments "make sense"when juxtaposed. Thus triplets of the form TTT are not necessarily inadmissible.Backward reading of the coding units (a type of error not prevented by the comma-lesscode) would only produce irrelevant transient interactions, since the correct polarityfor peptide-bond formation is established by the growing chain itself.

On the other hand, chain growth imposes a new requirement. Since a proteincontains many residues of the same kind as its Nvterminal amino acid, a mechanismis needed for distinguishing the N-terminallocus, so that a unique process can beinitiated there. This process is the attachment of the amino acid in such a way thatits - COOH will form a peptide bond with the next amino acid, whereas peptidebonds are not formed prematurely between other adjacent amino acids duringtheir transient occupancy of loci elsewhere on the template.

One way for an enzymic mechanism of peptide-bond formation to recognize anamino acid in the N-terminal locus would be by means of the unique chemical groupat the corresponding end of the messenger-RNA molecule, namely, by the 3/. or5'-hydroxyl (possibly phosphorylated). The <x-amino group of the appropriate aminoacid might be fixed here in such a way as to initiate chain growth. If the mere presenceof a unique terminal group on the template is sufficient, then the coding problemis not further complicated.

Another method of denoting the N-terminallocus of the template might be by aspecial code symbol, in addition to the usual coding unit for the N-terminal aminoacid itself. Transfer-RNA (tRNA) would then have to be equipped to recognize thespecial symbol; and since, presumably, any amino acid may be represented in the

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N-terminal position of some protein, all 20 species of tRNA would have to be soequipped. It is attractive to suppose that the ACC triplet by which all amino acidsare attached to their respective tRNA molecules may be such a recognition unit.

A third method for designating the N-terminallocus would be to specify a longenough sequence of adjacent residues to preclude any confusion with internal locifor the same amino acid. From the standpoint of its transfer function alone, thetRNA molecule seems unnecessarily long. However, as a means of recognizing withcertainty a particular sequence of coding units at the end of the template where chaingrowth is to begin, the information it contains may not be too redundant. tRNAcontains about 30 nucleotide triplets, and if it is largely folded back on itself, about15 base-pair triplets. This hypothesis predicts the existence of not one but a varietyof tRNA molecules corresponding to each of the 20 amino acids, since any particularamino acid would be followed by a different sequence of neighbors in the variousproteins in which it occupied the N-terminal position. Some explanation is thereforesuggested for the finding (Berg & Lagerkvist, 1961) that a given amino acid may becarried by more than one kind of tRNA molecule.

Of the three proposed methods of coding the N-terminal loci, the third is renderedunlikely by recent experimental evidence. It seems highly improbable that anyE. coli protein would contain a long N-terminal sequence identical to that of hemo­globin, yet RNA from E. coli is reported to function in the synthesis of hemoglobinby rabbit reticulocyte ribosomes (von Ehrenstein & Lipmann, 1961).

By whatever method chain growth is initiated, tRNA may be supposed to play animportant part. However, the special character of the attachment of the N-terminalresidue raises the question whether tRNA also mediates the insertion of aminoacids into internal positions. This question cannot yet be answered unequivocally.von Ehrenstein & Lipmann (1961) did show internal incorporation of leucine fromleucyl-tRNA into hemoglobin by reticulocyte microsomes, and that this leucine wasnot in equilibrium with external free leucine. However, transfer of leucine to anotheracceptor in the miorosomes, prior to incorporation, may not be out of the question.Nathans & Lipmann (19.61) showed that leucine was transferred from leucyl-tRNAto E. coli ribosomes, whereas free leucine and other amino acids were not incorporated.But in this case the position 'of the leucine in the ribosomal protein was not established.

I am indebted to Paul Berg for helpful discussions of this problem. Pertinentinvestigations in this laboratory are supported by grant CY-2797 from the NationalCancer Institute.

Department of PharmacologyStanford University School of MedicinePalo Alto, California, U.S.A.

Received 2 November 1961

AVRAM GOLDSTEIN

REFERENCESBerg, P. & Lagerkvist, U. (1961). Proceedings of the International Colloquium on Ribonucleic

Acids and Polyphosphates, Strasbourg.Bishop, J., Leahy, J. & Schweet, R. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1030.Crick, F. H. C., Griffith, J. S. & Orgel, L. E. (1957). Proc, Nat. Acad. Sci., Wash. 43, 416.Dintzis, H. M. (1961). Proc. Nat. Acad. Sci., Wash. 47, 247.von Ehrenstein, G. & Lipmann, F. (1961). Proe. Nat. Acad. Sci., Wash. 47, 941.Goldstein, A. (1961). Biochim. biophys. Acta, 53, 438.Nathans, D. & Lipmann, F. (1961). Proc, Nat. Acad. Sei., Wash. 47, 497.Steinberg, D., Vaughan, M. & Anfinsen, C. B. (1956). Science, 124, 389.