Functional analysis of transcription of the Mycobacterium tuberculosis 16S rDNA-encoding gene

Abstract

A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study. RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth. Transcription start points (tsp) were mapped by primer extension analysis of RNA from M. tuberculosis H37Rv and M. tuberculosis H37Ra. An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M. tuberculosis H37Rv and H37Ra. One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter. The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E. coli. The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene (cat) in a shuttle plasmid vector, pSD7. The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M. smegmatis. The M. smegmatis transformant exhibited a CAT activity of 16669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength. Two tsp utilized in M. tuberculosis were also employed in M. smegmatis. The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.