Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus

Abstract

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules.Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.The bacterial 50S ribosomal subunit is generally composed of a 23S and a 5S rRNA molecule and ribosomal proteins. However, some bacterial species have ribosomes that do not contain an intact 23S rRNA species. In vivo fragmentation of 23S rRNA has been reported for some cyanobacteria (10), for Agrobacterium tumefaciens (38), for Bdellovibrio bacteriovorus (34), for Salmonella species (5, 43), and for the closely related bacteria Rhodobacter sphaeroides (33), Rhodobacter capsulatus (28), and Paracoccus denitrificans (29). The biological significance of 23S rRNA fragmentation is not understood.Bacterial rRNA operons are transcribed into large precursor molecules often including tRNA sequences (23). These rRNA precursors are subsequently processed into the 23S, 16S, and 5S rRNA species. In Escherichia coli, two endoribonucleases that are involved in the processing of rRNA have been identified: RNase III and RNase E. RNase III cleaves doublestranded RNA regions with little sequence specificity (36,37) and is responsible for the generation of 23S and 16S rRNA precursor molecules from the larger 30S precursor (4, 44). RNase III is also responsible for the excision of intervening sequences from the rRNA of Salmonella species, resulting in fragmentation of the 23S rRNA (5). The enzymes involved in the final maturation of 16S and 23S rRNA from pre-16S and pre-23S rRNA have not been characterized; however, these reactions are most likely catalyzed by exoribonucleases (23,39). 5S rRNA is generated by cleavage of a 9S rRNA precursor that is catalyzed by the endoribonuclease RNase E (35). From comparison of different RNase E cleavage sites, the consensus sequence RAUUW (R = A or G; W = A or U) (14) has been postulated. RNase E cleaves single-stranded RNA regions with the maximal rate, when the cleavage site is preceded or followed by an mRNA hairpin-loop structure (14) (20), which is present in four copies on a single chromosome (15). The organization of the R. sphaeroides rrn operons is identical to the organization found in E. coli, with two tRNA genes in the 16...

Discordant Phylogenies within the rrn Loci of Rhizobia

“…The primers 450 and 1440 (66) were used to amplify the ITS region (66) and are located in conserved regions of the 3Ј end of the 16S rRNA gene and the 5Ј end of the 23S rRNA gene. The PCR products generated with this primer pair also contained the intervening region (16,27,34,36,38,53,54,71) of the 23S rRNA gene and permitted sequencing analysis of the 5Ј ends of the 23S rRNA genes by using primers 1431, 1432, 1439, and 1440 (66). The primer pair 1432 and 23S lowerB were used to amplify most of the 23S rRNA genes.…”

RNase III Processing of Intervening Sequences Found in Helix 9 of 23S rRNA in the Alpha Subclass of Proteobacteria

“…In alpha-Proteobacteria additional fragmentation sites were found. In Rhodobacter capsulatus and Rhodobacter sphaeroides, the 23S rRNA is fragmented at position 1200 (helix 46; E. coli numbering) due to IVS processing (16,27). In domain III of 23S rRNA of some Rhizobium and Agrobacterium strains sporadic fragmentation without the involvement of IVSs has been described (28,30).…”

Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence.

“…The primers (450 and 1440) used to amplify the ITS region (Table 2) are located in conserved regions of the 3h end of the 16S rRNA gene and the 5h end of the 23S rRNA gene. The PCR products generated with this primer pair also contained the intervening region Kordes et al, 1994 ;Lessie, 1965 ;Mackay et al, 1979 ;Marrs & Kaplan, 1970 ;Schuch & Loening, 1975 were used for sequencing the purified PCR products as described previously (van Berkum et al, 1996). The primer sequences used to sequence the ITS region are listed in Table 2.…”