Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

Abstract

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se, was not increased. Rather, these mutations appear to increase expression of the tnp'-'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1-3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification, importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3-5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

“…In particular, IF3 has been shown to play a significant role in start codon selection. 32,33,34 To examine the possibility that the hyperactive phenotype produced by mutations at 966 and 967 is due to an effect on IF3 binding, we cloned IF1, IF2 and IF3 into a pACYC177 35 derivative constructed in our lab, pKAN6, and placed them under control of the inducible araBAD promoter. 36, 37 As a negative control, we also measured the effect of initiation factor over-expression on GFP translation by ribosomes containing the G693U mutation in 16S rRNA.…”

Functional importance of RNA interactions in selection of translation initiation codons

“…Start-site selection of bacterial mRNAs includes a second type of RNA base pairing between the start codon and the anticodon loop of the initiator fMet-tRNA fMet , the fidelity of which is controlled by the translation initiation factor IF3 (Sacerdot et al, 1996;Sussman et al, 1996). The specificity of this interaction is also governed by initiation factor IF2 which recognizes the initiator tRNA and probably helps to correctly position it near the ribosomal decoding centre (Voorma, 1996).…”

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

“…Even though binding of IF1 to the ribosomal A-site could suggest that IF1 takes part in an initiation fidelity function [20,21], this hypothesis has not been tested so far (latest review [1]). In this study, we have investigated the relationship between IF1 and the nature of the +2 codon immediately following the initiation codon, thus representing the ribosomal Asite.…”