Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria

Abstract

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.

Atypical Processing in Domain III of 23S rRNA of Rhizobium leguminosarum ATCC 10004 T at a Position Homologous to an rRNA Fragmentation Site in Protozoa

“…It was reported that in some ␣-Proteobacteria, in addition to the RNase III cleavage in helix 9, helix 10 processing by an unknown enzyme(s) creates the 5Ј end of the 2.6-kb rRNA fragment (9,34,35). This prompted us to examine the extent of 23S rRNA sequences removed during the processing in the helix 9 region of R. leguminosarum.…”

“…Sequencing reactions of templates with known sequences were used as a nucleotide ladder (not shown). Thus, in addition to the RNase III cleavage of the IVS in helix 9, further processing events remove the helix 10 sequences from the primary 23S rRNA transcript in R. leguminosarum, as in other ␣-Proteobacteria (34,35).…”

“…No 23S rRNA processing was observed in an RNase III-deficient R. capsulatus mutant, showing that the failure of RNase III to cleave helix 9 also abolishes helix 10 processing in this organism. On the other hand, RNase III is not responsible for helix 10 cleavage, as shown by in vitro assays of substrates containing both helices (9,34,35).…”

“…The samples were ethanol precipitated, and aliquots were analyzed on a polyacrylamide-urea gel. [␣- 35 S]dATP-labeled sequencing reactions (T7 Sequencing Kit; Pharmacia Biotech) of the cloned DNA template were loaded on the same gel to map the positions of the primer extension signals.…”

Precise characterization of rDNA genes by intraspecies and inter-loci comparison of rDNA sequences and biochemical analysis of ribosomal RNA molecules in Agrobacterium tumefaciens

“…Recently, it has been reported that IVS is also present in two a -Proteobacteria members: Brucella melitensis (Bricker, 2000) and Rhizobium species (Selenska-Pobell and Evguenieva-Hackenberg, 1995). The IVS segment is removed, and further fragmentation occurs during rRNA processing (Zahn et al, 1999;Zahn et al, 2000;Zahn et al, 2001). The diversity of rRNA and rDNA described above has prompted extensive characterization and comparison.…”