The involvement of the anticodon adjacent modified nucleoside N-[9-( -D-ribofuranosyl) purine-6-ylcarbamoyl]-threonine in the biological function of E.coli tRNAile

Abstract

tRNAile was isolated from E. coli Cp 79 (leu-, arg-, thr-, his-, thiamin-, RCrel) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(beta-D-ribofuranosyl)- purin-6-ylcarbamoyl]threonine, t6Ado, than tRNAile from cells grown on an optimum concentration of thr and containing a normal complement of t6Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3'-terminal dinucleotide, and to form an ile-tRNAile-Tu-GTP complex. In contrast, the t6Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t6Ado-deficient ile-tRNA demonstrated no binding at Mg2+ concentrations less than or equal to 10 mM, while the normal ile-tRNA bound at low Mg2+ concentrations. Tetracycline had the same effect on the normal as on the t6Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t6Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t6Ado is required for proper codon-anticodon interaction.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…The hypermodified nucleoside N 6 -threonylcarbamoyladenosine (t 6 A 37 ) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site.…”

“…This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t 6 A 37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t 6 A 37 modification: TsaC, TsaD, TsaB, and TsaE.…”

“…The biosynthetic pathway of t 6 A 37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t 6 A 37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families.…”

“…Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t 6 A 37 , the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP.…”

“…This stabilization is necessary to over-come the low enthalpy of binding for U-A base pairs and tRNA Lys UUU in particular with three U-A base pairs (11). The size and location of t 6 A 37 on the Watson-Crick face of the nucleoside negate intra-loop base pairing with the invariant U33, which is critical for the U-turn backbone structure of tRNA.…”