Expression of the mcrA gene of escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region

Abstract

The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA+/Rg1A+ phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA+/Rg1A+ phenotype and directed production of a 23-kDa polypeptide. Smaller inserts displayed McrA-/Rg1A- phenotypes, though a 0.7-kb insert did direct production of a 24-kDa polypeptide. A construct carrying the 1.0-kb mcrA insert yielded a single 1.3-kb transcript. The mcrA transcript was found to start from C, G, T and G, namely the fourth, fifth, sixth and seventh nucleotides (nt), respectively, downstream from the last nt of the putative - 10 region. Two mcrA transcriptional/transational fusions were made in the pT7-7 expression vector and the protein encoded by these constructs were analysed. Regulation of mcrA expression was studied by quantitative Northern analysis of RNA from various mcrA clones. Together with a computer analysis of the translation initiation region in these mRNAs, the results suggest that the expression of mcrA may be regulated at the translational level. © 1995.