The Tn10-encoded tetracycline resistance mRNA contains a translational silencer in the 5' nontranslated region.

We performed a mutational analysis of the left half of Tn10-encoded tet operator O2, located in the 5' nontranslated region of the mRNA for the resistance protein TetA, and determined the importance of that region for translation efficiency and mRNA stability. Transcriptional fusions of 17 mutants to lacZ expressed the same amounts of beta-galactosidase, while translational fusions varied 35-fold in expression efficiency. The mRNA half-lives varied 24-fold, with 9.6 min for the most highly expressed mRNA and 0.4 min for the least efficiently expressed mRNA. Toeprint experiments were performed to distinguish whether these mutations define a determinant of mRNA stability or influence translation initiation. The highly expressed mRNA was 24-fold more efficient in forming the initiation complex in vitro than the low-expression mutant. It was concluded that this sequence, albeit located upstream of the ribosome-binding sequence, is an important determinant for efficient initiation of translation. Secondary-structure calculations of the mRNAs revealed no correlation of the potential to form double strands masking the ribosome-binding sequence with expression efficiency.

Lack of a 5' non-coding region in Tn1721 encoded tetR mRNA is associated with a low efficiency of translation and a short half-life in Escherichia coli.

The repressor-encoding tetR gene from Tn1721 is expressed with a very low efficiency. Its mRNA lacks an untranslated leader sequence. We have constructed protein fusions with the lacZ gene which contain between 14 and 157 5' nucleotides from the tetR gene. Since they are all expressed with similar efficiencies we conclude that the sequence information for initiation of translation is contained within the first 14 bases of the tetR coding region. These fusion transcripts are about 20-fold less efficiently translated than the wild type lacZ transcript. A toeprint analysis confirms that the initiation complex is indistinguishable from those formed by regular transcripts with 5' untranslated regions but occurs in a very low amount in vitro. Thus, the absence of a 5' leader causes a poor rate of translation initiation. The half-lives of tetR and tetR-lacZ mRNAs are about 30 seconds, which is 3-times lower than that of the wt lacZ mRNA. Inactivation of the ams/rne locus in E. coli stabilizes the tetR transcript more than ten-fold. The influence of translation on the tetR half-life is discussed.