Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16s rRNA coding region and continuing to the start of the 23s rRNA coding region. The 16s rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia culi. The regions of similarity include a -35 box, a -10 box, a stringent response element, antitermination signals, potential RNAase I11 processing sites and features of precursor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 1 6 s rRNA were also investigated. Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. Zeprae; although the principal features are common to both species there are notable differences.

Slow-growing mycobacteria have a single ribosomal RNA (rrn) operon, with the genes for 165, 235 and 55 rRNA being present in that order. The transcription start site of the rrn operon of Mycobacterium tuberculosis was identified in Escherichia coli. PCR methodology was used to amplify parts of the rrn operon, namely the leader region and the spacer-I region separating the 16s rRNA and 235 rRNA genes of Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium intracellulare, 'Mycobacterium lufu ' , Mycobacterium simiae and Mycobacterium marinum. The amplified DNA was sequenced. The sequence data, together with those obtained previously for Mycobacterium leprae and M. tuberculosis, were used to identify putative antitermination signals and RNase 111 processing sites within the leader region. Notable features include a highly conserved Box B element and a sequence of 31 nucleotides which is common to all eight slow-growers which were scrutinized. A secondary structure for mycobacterial precursor-I 65 rRNA was devised, based on sequence homologies and homologous nucleotide substitutions. The 18 nucleotides a t the 5'-end of spacer-I have the capacity of binding sequences close to the 5'-and 3'-ends of mature 16s rRNA, suggesting that secondary structure is important to the maturation process. All the slowgrowers, including M. leprae, conform to the same scheme of secondary structure. The scheme proposed for M. tuberculosis is a variant of the main theme. The leader and spacer sequences may prove a useful supplement to 165 rRNA sequences in establishing phylogenetic relationships between very closely related species. 'M. lufu'appears t o be a close relative of M. intracellulare.

The two ribosomal RNA (rrn) operons (rrnA and rrnB) of Mycobacterium smegmatis were investigated. The leader regions, part of the 16s rRNA genes, the spacer-I regions, part of the 235 rRNA genes, and the spacer-2 regions were amplified by PCR or by inverse PCR and the products were cloned and sequenced. No differences in the sequences of the two operons were detected downstream from the Box A antitermination element of the leader region.Upstream from Box A a slow-grower-like Box B antitermination element was found in rrnA but not in rrnB. Primer extension experiments revealed that the start of transcription lies a t least 370 nucleotides upstream from the 5'-end of the 16s rRNA gene and an RNase processing site near to the Box A element. Secondary structures were deduced for pre-16s rRNA and pre-23s rRNA which are distinct from, but closely related to, the corresponding structures of slowgrowing mycobacteria. On the basis of these results it is proposed that the emergence of the slow-growers from the main mycobacterial line was coincident with the deletion of a segment of DNA spanning an rmB-like operon, leaving an rmA-like operon as the sole source of rRNA. An explanation is also proposed for the need for two Box A motifs in the transcription of an rrn operon based on competition between the polymerase and the nascent 305 subunit for either protein 510 and/or Box A sequences.

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 rnyco bacter ia Yuan- The single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 165, 235 and 55 rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 165 rRNA and 235 rRNA genes and the spacer-2 region separating the 235 rRNA and 55 rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, 'Mycobacterium lufu ' and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 55 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 111 processing sites within the spacer4 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 235 rRNA and for precursor 55 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 55 rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 165 rRNA sequences in establishing phylogenetic relationships between very closely related species. The EMBL accession numbers for the nucleotide sequence data reported in this paper are X74056X74063 and X75599-X75602. (Shepard, 1960; K'inder & Rooney, 1970). Although ribosome biosynthesis is essential to cell proliferation, the strategy (or strategies) used by slow-growing mycobacteria to control the production of new ribosomes is not known. One facet of this strategy is the regulation of the biosynthesis of rRNA. The slow-growing mycobacteria have a single rRNA (rm) operon (Bercovier et a/., 1986) comprising genes for the rRNA family in the order 16s rRNA, 23s rRNA and 5s rRNA (see Fig. 1). The operon is transcribed as a single unit (precursor rRNA or prerRNA) which is cleaved by RNases to precursors of 16s rRNA (pre-16s rRNA), 23s rRNA (pre-23s rRNA) and Keywords

No abstract