In vivo study of Escherichia coli tryptophan tRNA identity

Abstract

The structural features determining the identity of Escherichia coli tRNA{dollar}\sp{lcub}\rm Trp{rcub}{dollar} have been studied in vivo. The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using an assay based on initiation of protein synthesis by mischarged mutants of the E. coli initiator tRNA. Substitution of the methionine anticodon, CAU, with the tryptophan anticodon, CCA, caused tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} to be aminoacylated with both methionine and tryptophan in vivo. This was established by sequence analysis of a reporter protein whose synthesis was initiated by a tryptophan codon. Conversion of the discriminator base of tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}(CCA) from A{dollar}\sb{lcub}73{rcub}{dollar} to G{dollar}\sb{lcub}73{rcub},{dollar} the base present in tRNA{dollar}\sp{lcub}\rm Trp{rcub},{dollar} eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of a tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar} derivative containing the tryptophan anticodon and discriminator base (tRNA{dollar}\sp{lcub}\rm fMet{rcub}{dollar}(CCA)/G{dollar}\sb{lcub}73{rcub}){dollar} to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion. Thus, all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G{dollar}\sb{lcub}73{rcub}{dollar} in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA{dollar}\sp{lcub}\rm Trp{rcub}.{dollar} Substitution of G{dollar}\sb{lcub}73{rcub}{dollar} with A{dollar}\sb{lcub}73{rcub},{dollar} C{dollar}\sb{lcub}73{rcub}{dollar} or U{dollar}\sb{lcub}73{rcub}{dollar} resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein.;In addition, the role of amino acid acceptor stem base pairs in determining the identity of tRNA{dollar}\sp{lcub}\rm Trp{rcub}{dollar} was studied using opal suppressor derivatives of tRNA{dollar}\sp{lcub}\rm Trp{rcub}{dollar}(UCA). Base pair substitutions in the first three positions of the acceptor stem caused 2- to 12-fold reductions in the efficiency of suppression without loss of the tryptophan specificity of the tRNA. When introduced in the context of wild-type tRNA{dollar}\sp{lcub}\rm Trp{rcub},{dollar} all of the acceptor stem derivatives were able to complement an E. coli strain harboring a temperature-sensitive tRNA{dollar}\sp{lcub}\rm Trp{rcub}{dollar} gene {dollar}(trpT\sb{lcub}\rm ts{rcub}){dollar} at the nonpermissive temperature (42{dollar}\sp\circ{dollar}C). However, three derivatives {dollar}\rm(tRNA\sp{lcub}Trp{rcub}/C\sb1{lcub}\cdot{rcub} G\sb{lcub}72{rcub},\ tRNA\sp{lcub}Trp{rcub}/C\sb2{lcub}\cdot{rcub} G\sb{lcub}71{rcub},\ tRNA\sp{lcub}Trp{rcub}/A\sb3{lcub}\cdot{rcub} U\sb{lcub}70{rcub}){dollar} required overexpression for growth at 42{dollar}\sp\circ{dollar}C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRNAs appear to be defective in translation. The results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA{dollar}\sp{lcub}\rm Trp{rcub}.{dollar}.;Substitutions of the {dollar}\rm C\sb1{lcub}\cdot{rcub} A\sb{lcub}72{rcub}{dollar} base pair in a methionine initiator tRNA containing a tryptophan anticodon and discriminator base {dollar}\rm(tRNA\sp{lcub}fMet{rcub}(CCA)/G\sb{lcub}73{rcub}){dollar} with a {dollar}\rm A\sb1{lcub}\cdot{rcub} U\sb{lcub}72{rcub},{dollar} the base pair found in tRNA{dollar}\sp{lcub}\rm Trp{rcub},{dollar} or a {dollar}\rm G\sb1{lcub}\cdot{rcub} C\sb{lcub}72{rcub}{dollar} resulted in conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo. However, changes to {dollar}\rm U\sb1{lcub}\cdot{rcub} A\sb{lcub}72{rcub}{dollar} or {dollar}\rm C\sb1{lcub}\cdot{rcub} G\sb{lcub}72{rcub}{dollar} in the {dollar}\rm tRNA\sp{lcub}fMet{rcub}(CCA)/G\sb{lcub}73{rcub}{dollar} resulted in misaminoacylation and/or defects in translation. These data indicate that the {dollar}\rm A\sb1{lcub}\cdot{rcub} U\sb{lcub}72{rcub}{dollar} base pair is a context dependent, negative identity element of tRNA{dollar}\sp{lcub}\rm Trp{rcub}.{dollar}.