Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression

Abstract

To test features of the current model of transcription attenuation in amino acid biosynthetic operons, alterations were introduced into the trp operon leader region and expression of the mutated operons was examined in miaA and miaA+ Escherichia coli strains that lacked the trp repressor. The miaA mutation prevents modification of the adenosine residue immediately 3' of the anticodon of tRNAs that interact with codons beginning with uridine. The undermodified tRNA(Trp) in miaA strains is thought to increase readthrough at the trp attenuator by slowing ribosome movement over two tandem Trp codons in the 14-codon leader peptide coding region. The rate of translation of these two "control codons" is thought to be the key step in determining the extent of transcription attenuation in the trp leader region. Sequential deletion of trpL DNA specifying the leader peptide initiation region, RNA segment 1, RNA segment 2 and RNA segment 3 alternately decreased and increased trp operon expression, a result consistent with previous findings in another bacterium and the generally accepted model for transcription attenuation. Replacement of the tandem Trp control codons by AGG-UGC (Arg-Cys) codons eliminated the miaA-dependent increase in transcription readthrough. Replacement of the Trp control codons by AGG-UGA (Arg-stop) codons caused complete readthrough at the trp attenuator as well as abolishing the miaA effect. Presumably, the ribosome terminating translation at the new UGA codon mimics the effect of a stalled ribosome at the Trp control codons. This finding suggests that ribosome dissociation at some stop codons is slow relative to the time required for transcription of the trp leader region. Thus, most ribosomes translating the trp leader peptide coding region may remain attached to the natural UGA stop codon until after the attenuation decision is made. The interpretation supports models for trp operon attenuation in which the elevated basal level readthrough is determined by occasional ribosome release prior to synthesis of the 3:4 terminator hairpin.