Structural requirements for recognition of Escherichia coli initiator and non-initiator transfer ribonucleic acids by bacterial T factor.

Escherichia co2i formylmethionine tRNA is unable to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. Treatment of tRNAfMet with sodium bisulfite produces cytidine to uridine base changes in the tRNA structure and greatly enhances its binding affinity for T factor. The ternary complex formed with the bisulfite-modified initiator tRNA has properties analogous to those of EF-Tu . GTP ‘AA-tRNA complexes formed with non-initiator tRNAs. Enzymatic formylation of modified Met-tRNArMet completely eliminates its ability to bind to EF-Tu. Unmodified tRNAfMet is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5’ terminus. Bisulfite-catalyzed conversion of this unpaired @dine to uridine results in formation of a normal uridine-adenosine base pair at the end of the acceptor stem. In order to determine whether this modification affects T factor recognition, partially modified Met-tRNA fM e t was labeled with azP at the 5’ terminus and the fraction capable of forming an EF-Tu GTP . Met-tRNA fMe t complex was separated from unbound Met-tRNA fMe t by Sephadex G-100 gel filtration. Terminal nucleotide analysis of the tRNA bound in the complex showed that it was greatly enriched in [5’-**P]uridylate compared to the starting tRNA. Removal of the 5’-phosphate from several normal AAtRNAs was found to drastically reduce the ability of these tRNAs to form stable ternary complexes with EF-Tu. The requirement for base pairing at the end of the acceptor stem therefore appears to result from a requirement for a specific spatial orientation of the 5’-terminal phosphate for T factor binding. Bisulfite modification of the 3’-terminal CpCpA-OH sequence of Met-tRNAfMet reduces its binding affinity for EF-Tu, indicating that this region of the molecule is also involved in formation of a stable ternary complex. Met-tRNArMet containing a fully base-paired acceptor stem