Analysis of the initiator tRNA genes from a slow-and a fast-growing mycobacterium

Abstract

Initiation of protein synthesis is a major post-transcriptional regulatory step in gene expression. The initiator tRNA gene from Mycobacterium smegmatis, a fast-growing mycobacterium, was characterized and compared with its counterpart from Mycobacterium tuberculosis, a slow-growing mycobacterium. In both mycobacteria, the functional initiator tRNA genes were found in a single copy. Unlike the M. tuberculosis initiator tRNA, the CCA end of the M. smegmatis initiator is not encoded in the gene, and it is most likely added post-transcriptionally. Transcription start site mapping allowed accurate assignment of the hexameric −l10 and −35 promoter elements for both genes. These elements of the M. smegmatis initiator tRNA gene contain single nucleotide changes compared to their respective counterparts in the M. tuberculosis gene. Chloramphenicol acetyl transferase reporter assays suggested that the promoter of the initiator tRNA gene from M. smegmatis is twice as strong as that of M. tuberculosis, irrespective of whether the assays were performed in the fast-growing homologous host (M. smegmatis) or the slow-growing heterologous host (M. tuberculosis). Characterization of the M. smegmatis metU promoter, in this study, provides a valuable tool for the expression of genes in mycobacteria.