Biological Activity of Escherichia coli tRNAPhe Modified in Its C‐C‐A Terminus

Jacov Tal, Uriel Z. Littauer, Murray P. Deutscher

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43 Scopus citations


An improved method for the stepwise degradation of tRNA by means of periodate oxidation, amine‐catalyzed elimination and alkaline phosphatase treatment was developed. Alkaline phosphatase was found to hydrolyze the exposed 3′‐phosphate group of tRNA…N‐C‐Cp at a much faster rate than that of tRNA…N‐Cp. This difference in rate of dephosphorylation was manifested at several temperatures. Terminally modified Escherichia coli tRNA, from which all or part of the C‐C‐A was removed was characterized by its ability to accept AMP and CMP using rat liver tRNA nucleotidyltransferase. It was found that tRNA…N‐C‐C accepts AMP but not CMP; tRNA…N‐C accepts CMP and AMP in a molar ratio of 1:1 and tRNA…N accepts CMP and AMP in a molar ratio of 2:1. In addition, it was observed that tRNA…N‐C accepts AMP in the absence of CTP in the reaction mixture. The product of AMP incorporation into tRNAPhe…N‐C was shown to be tRNAPhe…‐N‐C‐A, by electrophoretic analysis of the 3′‐terminal fragment obtained by T1 ribonuclease digestion. It was also shown that tRNA…N‐C‐A can not be acylated with a mixture of 15 amino acids, suggesting that an intact C‐C‐A sequence is an absolute requirement for aminoacylation. Under conditions of high enzyme concentrations, in vitro, tRNA nucleotidyltransferase also misincorporates AMP residues into tRNA…N. The ability of various terminally modified tRNAs to inhibit phenylalanine charging of tRNA was determined by kinetic measurements. tRNAox, obtained by periodate oxidation of tRNA, served as a competitive inhibitor for phenylalanine acylation of intact tRNA. The measured Ki value of 40 nM equaled the apparent Km for tRNAPhe. tRNA…N‐C‐Cp, tRNA…N‐C‐C, tRNA…N‐C, tRNA…N and tRNA…N‐C‐A were found to be less competent competitive inhibitors, their Ki values being one order of magnitude higher. These results suggest that the terminal adenosine participates in the binding of tRNAPhe to phenylalanyl‐tRNA synthetase and that the relative position of this adenosine with respect to the rest of the tRNA molecule is probably critical for an effective binding to the enzyme. The reduction of tRNAox with sodium borohydride converts the terminal dialdehyde group to a dialcohol lacking a covalent bond between the C′2 and C′3 of the terminal adenosine. Whereas unfractionated tRNAox could not serve as an acceptor for any amino acids, reduction to tRNAox‐red restored the abilities to accept phenylalanine, methionine and tyrosine, On the other hand, acceptor activities for arginine, isoleucine, aspartic acid, histidine, serine, tryptophan and valine were not regained. It is concluded that the covalent bond between the C′2 and C′3 of the terminal adenosine is a decisive determinant for the aminoacylation of some Escherichia coli tRNAs but plays a minor role in other species. Terminally modified tRNAs inhibited poly(U)‐directed [14C]phenylalanyl‐tRNA binding to 30‐S or a mixture of 30‐S and 50‐S ribosomes as effectively as intact, unacylated tRNA. Thus, the C‐C‐A terminus does not contribute significantly to the binding of tRNAPhe to the poly(U) · ribosome complex. tRNA…N‐[14C]C was incubated with tRNA nucleotidyltransferase in the absence of nucleoside triphosphates and then hydrolyzed by alkali. The ratios of cytidine to cytidylic acid obtained before and after incubation were found to be identical. Thus, tRNA nucleotidyltransferase does not carry out transnucleotidation at the 3′‐terminus of tRNA…N‐C. The mode by which tRNA nucleotidyltransferase adds CMP to tRNA…N, was examined. The results suggest a “nonprocessive” mechanism, i.e. the enzyme dissociates from the substrate tRNA after the addition of one mononucleotide residue and then randomly attaches to another tRNA chain.

Original languageEnglish
Pages (from-to)478-491
Number of pages14
JournalEuropean Journal of Biochemistry
Issue number4
StatePublished - 1 Jan 1972
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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