Structural Requirements for Recognition of Escherichia coli Initiator and Non-Initiator Transfer Ribonucleic Acids by Bacterial T Factor

Escherichia coli 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-tRNAfMet 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 cytidine 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-tRNAfMet was labeled with 32P at the 5' terminus and the fraction capable of forming an EF-Tu·GTP·Met-tRNAfMet complex was separated from unbound Met-tRNAfMet by Sephadex G-100 gel filtration. Terminal nucleotide analysis of the tRNA bound in the complex showed that it was greatly enriched in [5'-32P]uridylate compared to the starting tRNA. Removal of the 5'-phosphate from several normal AA-tRNAs 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-tRNAfMet containing a fully base-paired acceptor stem and normal CpCpA-OH sequence can be completely bound as EF-Tu·GTP·Met-tRNAfMet in the presence of excess T factor, however this ternary complex is less stable than the complexes formed with Met-tRNAmMet, Phe-tRNAPhe, or Gln-tRNAGln, suggesting that other structural features outside of the 3'- and 5'-terminal regions may be involved in T factor recognition of AA-tRNAs.