No abstract

For still unknown reasons, the 23S rRNA of many ␣-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The ␣-proteobacterium Rhizobium leguminosarum ATCC 10004 T possesses two fragmentation sites in its 23S rRNA. The first one harbors an intervening sequence in helix 9 which is cleaved by RNase III. We demonstrate that the mature 5 end of the resulting 2.6-kb rRNA fragment is generated by additional removal of helix 10. A fraction of the 2.6-kb rRNA is further processed in domain III, giving rise to two 1.3-kb rRNA fragments. We mapped the domain III fragmentation site and found it to be at a position which has only been reported for trypanosomatid protozoa. This fragmentation site is also unique in that it lacks an intervening sequence. We found that the simultaneous occurrence of 2.6-kb and 1.3-kb rRNA fragments is not due to interoperonal sequence differences but rather reflects slow processing. The different characteristics of the two fragmentation sites in the 23S rRNA suggest that they are processed by different mechanisms. Interestingly, the amount of 2.6-kb rRNA varies during culture growth. We observed a transient increase in the relative amount of 2.6-kb rRNA fragments during the first hours after inoculation, which points to changes in the ratio of rRNA synthesis rate to domain III processing rate during the growth of a culture.Fragmentation of the RNA of the large ribosomal subunit has been described in many eukaryotic and prokaryotic organisms (5, 8, 9, 10, 11, 19, 24, 27-29, 32, 34). All 23S rRNA processing sites identified in Eubacteria (summarized in references 9 and 28) have until now been located at positions phylogenetically identical to rRNA fragmentation sites found in Eucarya (10, 11). This fact raises questions about the origin and evolution of the rRNA genes, and about the physiological implications of rRNA fragmentation. Additionally, the exact knowledge of the primary and secondary structure of the mature 23S rRNA fragments is important since 23S rRNA sequences are used as phylogenetic markers and for strain identification (6,16,30,31).The 23S rRNA of Rhizobium leguminosarum ATCC 10004 T possesses two processing sites (Fig. 1). The first one is at a position common to many ␣-proteobacterial species, in helix 9 of 23S rRNA. The helix 9 of R. leguminosarum contains an intervening sequence (IVS) (29) which is removed by RNase III cleavage (9). The 23S rRNA processing in this region leads to the occurrence of a short 5Ј rRNA fragment with a length of approximately 130 nucleotides and a 3Ј rRNA fragment with 2.6-kb length (27). Recently it has been shown that in Rhodopseudomonas palustris the RNase III cleavage of the IVS in helix 9 represents only an initial event of subsequent efficient processing steps leading to the removal of helix 10 and production of a 5.8S-like rRNA (34). The second processing site in 23S rRNA of R. leguminosarum ATCC 10004 T is located in the central regio...