Nucleotide sequences of the spacer-1, spacer-2 and trailer regions of the rrn operons and secondary structures of precursor 23S rRNAs and precursor 5S rRNAs of slow-growing mycobacteria.

Abstract

The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 16S, 23S and 5S rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 16S rRNA and 23S rRNA genes and the spacer-2 region separating the 23S rRNA and 5S rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, 'Mycobacterium lufu' and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 5S rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase III processing sites within the spacer-1 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 23S rRNA and for precursor 5S rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 5S rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species.