Ribosome-binding sites and RNA-processing sites in the transcript of the Escherichia coli unc operon

Abstract

The polycistronic mRNA encoding the nine genes of the unc operon of Escherichia coli was studied. We demonstrated the ribosome-binding capabilities of six of the nine unc genes, uncB, uncE, uncF, uncH, uncA, and uncD, by using the technique of primer extension inhibition or "toeprinting." No toeprint was detected for the other genes, uncI, uncG, and uncC. The lack of a toeprint for uncG suggests that this gene is expressed by some form of translational coupling, such that either uncG is read by ribosomes which have translated the preceding gene, uncA, or translation of uncA is required for ribosome binding at the uncG site. RNA sequencing and primer extension in the regions of uncI and uncC, the first and last genes in the operon, respectively, gave less intense signals than those obtained for the other unc genes. This suggested that there are fewer copies of those regions of the transcript and that processing of the unc transcript occurred. Using primer extension and RNA sequencing, we identified sites in the unc transcript at which processing appears to take place, including a site which may remove much of the uncI portion of the transcript. Northern (RNA) blot analysis of unc RNA is consistent with the presence of an RNA-processing site in the uncI region of the transcript and another in the uncH region. These processing events may account for some of the differential levels of expression of the unc genes.