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

• Published in: Gene 1995-05, Vol.157 (1), p.201-207
• Main Authors: Shivapriya, R., Prasad, Ranjan, Narayanan, Iyer Lakshmi, Krishnaswamy, S., Dharmalingam, K.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Base Sequence; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Frameshift Mutation; Gene Expression Regulation, Bacterial; Genes, Bacterial; Models, Structural; Molecular Sequence Data; Nucleic Acid Conformation; overexpression; Oxidoreductases; Phenotype; Plasmids; primer extension; regulation; Restriction Mapping; RNA, Bacterial - biosynthesis; RNA, Bacterial - isolation & purification; S1 analysis; Transcription, Genetic
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(94)00746-F

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.