Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.

We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively.     

Escherichia coli 23S ribosomal RNA truncated at its 5'' terminus.

In a strain of E. coli deficient in RNase III (ABL1), 23S rRNA has been shown to be present in incompletely processed form with extra nucleotides at both the 5' and 3' ends (King et al., 1984, Proc. Natl. Acad. Sci. U.S. 81, 185-188). RNA molecules with four different termini at the 5' end are observed in vivo, and are all found in polysomes. The shortest of these ("C3") is four nucleotides shorter than the accepted mature terminus. In growing cells of both wild-type and mutant strains up to 10% of the 23S rRNA chains contain the 5' C3 terminus. In stationary phase cells, the proportion of C3 termini remains the same in the wild-type cells; but C3 becomes the dominant terminus in the mutant. Species C3 is also one of the 5' termini of 23S rRNA generated in vitro from larger precursors by the action of purified RNase III. We therefore suggest that some form of RNase III may still exist in the mutant; and since no cleavage is detectable at any other RNase III-specific site, the remaining enzyme would have a particular affinity for the C3 cleavage site, especially in stationary phase cells. We raise the question whether the C3 terminus has a special role in cellular metabolism.     

The ordered processing of intervening sequences in 23S rRNA of Rhodobacter sphaeroides requires RNase J

The essential processing of ribosomal rRNA precursors requires concerted and sequential cleavages by different endo- and exoribonucleases. Despite long lasting investigations of these processes the exact order of steps remained elusive. Many bacteria perform additional rRNA processing steps by removing intervening sequences within the 23S rRNA. This leads to disintegration of the 23S rRNA and discontinuously assembled fragments within the ribosomes. The maturation of these fragments also requires successive cleavage events by different RNases. Our study reveals that the 5'-to-3' exoribonuclease RNase J is responsible for the final 5'-end maturation of all three 23S rRNA fragments in the α-proteobacterium Rhodobacter sphaeroides. Additionally the results show that 5'- and 3'-processing steps are closely coupled: mature 5'-ends are a strict prerequisite for the final 3'-trimming of the 23S rRNA fragments.     

Processing pathway of Escherichia coli 16S precursor rRNA.

Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at both 5' and 3' mature termini, with the concomitant release of precursor fragments which are further metabolized by both exo- and endonucleases. In wild-type cells rapid cleavages by RNase III in precursor-specific sequences precede the subsequent formation of the mature ends; mature termini can, however, be formed directly from pre-16S rRNA with no intermediate species. The direct maturation is most evident in a strain deficient in RNase III, and the results in whole cells are consistent with results from maturation reactions in vitro. Thus, maturation does not require cleavages within the double-stranded stems that enclose mature rRNA sequences in the pre-16S rRNA.     

Ordered processing of Escherichia coli 23S rRNA in vitro.

In an RNase III-deficient strain of E. coli 23S pre-rRNA accumulates unprocessed in 50S ribosomes and in polysomes. These ribosomes provide a substrate for the analysis of rRNA maturation in vitro. S1 nuclease protection analysis of the products obtained in in vitro processing reactions demonstrates that 23S rRNA processing is ordered. The double stranded stem of 23S rRNA is cleaved by RNase III in vitro to two intermediate RNAs at the 5' end and one at the 3' end. Mature termini are then produced by other enzyme(s) in a soluble protein fraction from wild-type cells. The nature of the reaction at the 5' end is not clear, but the reaction at the 3' end is exonucleolytic, producing three heterogeneous mature termini. The two reactions are coordinated; 3' end maturation progresses concurrently with cleavages at the 5' end. Two results suggest a possible link between final maturation and translation: in vitro, mature termini are formed efficiently in the presence of additives required for protein synthesis; and all the processing intermediates detected from in vitro reactions are also found in polysomes from wild-type cells.     

Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli.

23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331. RNase III is a double strand (ds)-specific endoribonuclease. This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein. Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site. Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.     

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

For still unknown reasons, the 23S rRNA of many alpha-Proteobacteria shows a unique fragmentation pattern compared to other bacteria. The 23S rRNA processing involves RNase III and additional, yet unidentified enzymes. The alpha-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.     

Sensing of 5' monophosphate by Escherichia coli RNase G can significantly enhance association with RNA and stimulate the decay of functional mRNA transcripts in vivo

The RNase E/G family of endoribonucleases has a central role in RNA degradation and processing. Previous work has shown that their cleavage of substrates in vitro can be stimulated by the presence of a 5' monophosphate group. It has not however, established the importance of this activation for any natural RNA processing or decay pathway in vivo. Here we provide for Escherichia coli RNase G the first evidence that the sensing of a 5' monophosphate is required in vivo for the normal rapid decay of functional mRNAs; moreover, we show in vitro that, in contrast to a previous study, the presence of a 5' monophosphate can enhance the affinity of RNase G binding to RNA. The implications of these results along with our finding that the maturation of 16S rRNA is unaffected in cells containing an RNase G mutant impaired in 5' end sensing are discussed with regard to current models of RNA processing and decay and the molecular mechanism that underlies RNA cleavage by the RNase E/G family.