Characterization of the ribosomal RNA operons of Haloarcula marismortui

The genome of Haloarcula marismortui contains two ribosomal RNA operons, designated as rrnA and rrnB (and possibly a third operon designated as rrnC) of which the characterization of the rrnA and rrnB operons are presented. Characterization of the rrnA and rrnB operons involved the analysis of primary and secondary structures and in vivo studies of the primary transcripts and processing intermediates. It was found that the gene orders of the rrnA and rrnB operons were 5-16S rRNA-tRNAA l a-23S rRNA-5S rRNA-tRNACys- 3' and 5-16S rRNA-23S rRNA-5SrRNA-3', respectively. Computing the substitution rates for the entire rrnA and rrnB operons demonstrated that the major differences are localized in the non coding regions, that is the regions including the 5'-flanking of the 16S rRNA, 16S-23S rRNA spacer and the 3'-flanking of the 5S rRNA gene. The percentage similarities between the 16S, 23S and 5S rRNAs of rrnA and rrnB are 95%, 98.7% and 98.3%, respectively. A pairwise sequence comparison between the 23S rRNA sequence of the rrnC operon (Brombach et al., 1989) and the other two operons, rrnA and rrnB, revealed that the sequence similarities are 98.8% and 99.6%, respectively. The 5S rRNA sequence from the rrnC operon is identical to the rrnA sequence. The nucleotide substitutions within the 16S rRNA genes of rrnA and rrnB operons are concentrated in three separate domains 58-321, 508-823 and 986-1158. About 60% of the substitutions are concentrated within the 508-823 domain and are compensatory, affecting both components of the nucleotide base pairs within defined rRNA helices. Using nuclease Sl protection assays, it was shown that the 16S rRNAs from the rrnA and rrnB operons are expressed and present in intact 70S ribosomes. A comparison of the 23S rRNAs from the rrnA and rrnB operons showed that the substitutions are located within the variable regions of domains I, m, IV and VI of the universal secondary structures of 23S rRNAs. The 5S rRNA sequences of the two operons differ at two nucleotide positions in the helix IV of the universal secondary structure for the 5S rRNA. The 5'-flarucing regions of the rrnA contains four tandem promoters whereas the rrnB operon contains a single tandem promoter and a second promoter-like sequence. An internal promoter sequence was present within the 16S-23S spacer regions of all three operons from Ha. marismortui. Putative secondary structures of the primary transcripts from the rrnA operon showed that the 16S and 23S rRNAs are surrounded by inverted repeat structures containing the "bulge-helix-bulge" motif which is recognized by a processing endonuclease. In the case of the rrnB operon, the inverted repeat structure surrounding the 23S rRNA is identical to that of the rrnA operon and processing follows the same pathway. However, the inverted repeat structure surrounding 16S rRNA from rrnB does not contains the "bulge-helix- bulge" motif and its processing follows a distinct pathway. The 16S rRNA processing of the rrnB occurs at a single position within the 5'-flanking region and at three positions within the 16S-23S spacer region. The nucleotides present in these cleavage sites and their surrounding regions showed no sequence conservation. The 5S rRNA processing occurs close to or at its 5'- and 3'-ends. Two apparent termination sites were detected for the rrnA operon and they were found to be located downstream of the tRNACys RNA. Phylogenetic analysis of the 508-823 region of the 16S rRNA, the entire 16S rRNA, 23S rRNA genes and the combination of 16S-23S-5S rRNA genes were performed using the PAUP program. The phylograms from the 16S rRNA, 23S rRNA and the 16S-23S-5S rRNA sequences indicated that the rrnA and rrnB group together and the rate of divergence of the rrnB operon is higher than that of the rrnA operon. However, comparison of the 508- 823 regions showed that the two operons do not group together and that rrnB is evolving slower than rrnA. Based on the comparisons made between the rrnA and rrnB operons, it is obvious that the rrnB operon is different from the rrnA operon in its gene order, rRNA sequences and 16S rRNA processing and also probably evolving at a different rate than the rrnA operon.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat