Abstract
Abstract 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.
Highlights
Escherichia co2iformylmethionine tRNA is unable to form a ternary complex with bacterial T factor and GTP, as measured by SephadexG-50 gel filtration
In order to determine whether this modification affects T factor recognition, partially modified Met-tRNA fMet was labeled with azPat the 5’ terminus and the fraction capable of forming an EF-Tu GTP .Met-tRNA fMet complex was separatedfrom unbound Met-tRNA fMet by Sephadex G-100 gel filtration
We have recently reported that following introduction of four cytidine to uridine base changes into the primary structure of E. coli formylmethionine tRNA, this tRNA is able to form a stable complex with T factor and GTP in a manner analogous to non-initiating tRNAs [7]
Summary
Escherichia co2iformylmethionine tRNA is unable to form a ternary complex with bacterial T factor and GTP, as measured by SephadexG-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
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