ESCHERICHIA-COLI TRANSLATION INITIATION-FACTOR 3 DISCRIMINATES THE INITIATION CODON IN-VIVO

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS10 transposase gene (tnp), we unexpe ctedly obtained viable mutants defective in translation initiation fac tor 3 (IF3), Several lines of evidence led us to conclude that transpo sase expression, per se, was not increased. Rather, these mutations ap pear to increase expression of the tnp'-'lacZ gene fusions used in thi s 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 m utations on initiation from various start codons, Beginning with an ef ficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These po tential start codons fall into distinct classes with regard to transla tion efficiency in vivo: Class I codons (AUG, GUG, and UUG) support ef ficient translation; Class IIA codons (CUG, AUU, AUG, AUA, and ACG) su pport translation at levels only 1-3% that of AUG; and Class IIB codon s (AGG and AAG) permit levels of translation too low for reliable quan tification. Importantly, the IF3 mutations had no effect on translatio n from Class I codons, but they increased translation from Class II co dons 3-5-fold, and this same effect was seen in other gene contexts, T herefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observati ons. We discuss these observations as they relate to IF3 autoregulatio n and the mechanism of IF3 function.