RNase E is involved in 5'-end 23S rRNA processing in alpha-Proteobacteria

In Rhodobacter capsulatus and Rhizobium leguminosarum, an internal transcribed spacer consisting of helices 9 and 10 is removed during 23S rRNA processing, which leads to the occurrence of a 5.8S-like rRNA. The particular rRNA maturation steps are not known, with exception of the initial RNase III cleavage in helix 9. We found that GC-rich stem-loop structures of helix 9, which are released by RNase III, are immediately degraded. The degradation of helix 10 is slower and its kinetics differs in both species. Nevertheless, the helix 10 processing mechanism is conserved and includes cleavages by RNase E.     

Mutations at position A960 of E. coli 23 S ribosomal RNA influence the structure of 5 S ribosomal RNA and the peptidyltransferase region of 23 S ribosomal RNA

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesis and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Escherichia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.     

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.     

5S ribosomal RNA is contained within a 25 S ribosomal RNA that accumulates in mutants of Escherichia coli defective in processing of ribosomal RNA.

A temperature-sensitive mutant strain of Escherichia coli defective in two RNA processing enzymes, RNase III and RNase E (rnc. rne), fails to produce normal levels of 23 S and 5 S rRNA at the non-permissive temperature. Instead, a molecule larger than 23 S is produced. This molecule, designated 25 S rRNA, can be processed in vitro to produce p5 rRNA. These findings further our understanding of the overall processing events of ribosomal RNA which take place in the bacterial cell.     

Mini-III, a fourth class of RNase III catalyses maturation of the Bacillus subtilis 23S ribosomal RNA

Ribonuclease III (RNase III) type of enzymes are double-stranded RNA (dsRNA)-specific endoribonucleases that have important roles in RNA maturation and mRNA decay. They are involved in processing precursors of ribosomal RNA (rRNA) in bacteria as well as precursors of short interfering RNAs (siRNAs) and microRNAs (miRNAs) in eukaryotes. RNase III proteins have been grouped in three major classes according to their domain organization. In this issue of Molecular Microbiology, Redko et al. identified a novel class of bacterial RNase III, named Mini-III, consisting only of the RNase III catalytic domain and functioning in the maturation of the 23S rRNA in Bacillus subtilis. Its absence from proteobacteria reveals that this step is mechanistically different from the corresponding step in Escherichia coli. The fact that Mini-III orthologues are present in unicellular photosynthetic eukaryotes and in plants opens new opportunities for functional studies of this type of RNases.     

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.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Extensions of the known sequences at the 3'' and 5'' ends of 23S ribosomal RNA from Escherichia coli, possible base pairing between these 23S RNA regions and 16S ribosomal RNA.

Extensions of the known sequences at both 3' and 5' ends of 23S ribosomal RNA are presented: The 5' terminal is pG-G-U-U-A-A-G-Cp or pG-G-U... G-U-U-A-A-G-Cp, with a very short sequence between Up and Gp and the 3'terminal is G-A-A-C-C-G-A-(G)-G-C-U-U-A-A-C-C-U-UOH. These two terminal regions exhibit a high degree of complementarity. In addition, extensive complementarities are also found between the 5'terminal sequence of 23S RNA and a sequence contained in section A of the 16S ribosomal RNA, and between the 3'terminal sequence of 23S RNA and sequences in sections O and J in the 16S RNA. The degree of complementarity between the two extremities of 23S RNA, and between these extremities and regions of the 16S RNA, is far greater than would be expected on a random basis suggesting a possible involvement of this base-pairing in the functioning of ribosomes. This possibility is discussed.     

Movement of the 3'-end of 16 S RNA towards S21 during activation of 30 S ribosomal subunits

Fluorescence techniques were used to study conformational changes that occur in inactive E. coli 30 S ribosomal subunits during activation by heating in 12 mM Mg2+. Activation is associated with movement of a fluorophore on the 3'-end of 16 S RNA into a less polar environment and towards a probe on the cysteine thiol of ribosomal protein S21. The conformational change causes an apparent decrease in distance between the probes from 59 to 52 A as determined by non-radiative energy transfer.