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.     

Mini-III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis

The late steps of both 16S and 5S ribosomal RNA maturation in the Gram-positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram-negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5' and 3' extremities of B. subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini-III contains an RNase III-like catalytic domain, but curiously lacks the double-stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well-known members of this family of enzymes. Cells lacking Mini-III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini-III functions much more efficiently on precursor 50S ribosomal subunits than naked pre-23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini-III recognizes and cleaves double-stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.     

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.     

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.     

Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively 'annealing' sequences around the 5' end and sequences around the 3' end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures.The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

The 3'' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.