Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.     

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.     

Co-evolution of tRNA 3' trailer sequences with 3' processing enzymes in bacteria

Maturation of the tRNA 3' terminus is a complicated process in bacteria. Usually, it is initiated by an endonucleolytic cleavage carried out by RNase E and Z in different bacteria. In Escherichia coli, RNase E cleaves AU-rich sequences downstream of tRNA, producing processing intermediates with a few extra residues at the 3' end; these are then removed by exoribonuclease trimming to generate the mature 3' end. Here we show that essentially all E. coli tRNA precursors contain a potential RNase E cleavage site, the AU-rich sequence element (AUE), in the 3' trailer. This suggests that RNase E cleavage and exonucleolytic trimming is a general pathway for tRNA maturation in this organism. Remarkably, the AUE immediately downstream of each tRNA is selectively conserved in bacteria having RNase E and tRNA-specific exoribonucleases, suggesting that this pathway for tRNA processing is also commonly used in these bacteria. Two types of RNase E-like proteins are identified in actinobacteria and the alpha-subdivision of proteobacteria. The tRNA 3' proximal AUE is conserved in bacteria with only one type of E-like protein. Selective conservation of the AUE is usually not observed in bacteria without RNase E. These results demonstrate a novel example of co-evolution of RNA sequences with processing activities.     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

Identification of a ribonuclease P-like activity from human KB cells.

An endoribonuclease which cleaves tRNA precursor molecules has been partially purified from human KB tissue culture cells. This activity is found in cytoplasmic fractions but is not detectable in the nucleoplasm. tRNA precursor molecules from both E. coli and KB cells are cleaved by this novel activity to produce 5' phosphate-terminated oligonucleotides. E coli RNAase P and the KB cell nuclease both make a single endonucleolytic scission in E. coli tRNATyr precursor, thereby separating the 41 extra nucleotides on the 5' end of the precursor molecule from the 5' terminal sequence of the mature tRNATyr molecule. The cleavage products generated from other E. coli tRNA precursors by the KB cell activity are identical in size to those produced by RNAase P. The KB cell endoribonuclease requires Mg2+ and a monovalent cation (Na+, K+, or NH4+) for function. The enzymatic activity has a broad pH optimum, centered near pH 8.0, and the activity is inhibited by tRNA. Several KB cell RNAs with long half-lives in vivo, including 5S and bulk 4S RNA, are not cleaved by this nuclease. The KB cell endoribonuclease resembles E. coli RNAase P in its substrate specificity, pH optimum, ion requirements, and sensitivity to tRNA. These properties and the cytoplasmic localization of the novel endoribonuclease indicate its involvement in the biosynthesis of KB cell tRNA.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

Crystal structure of the phosphorolytic exoribonuclease RNase PH from Bacillus subtilis and implications for its quaternary structure and tRNA binding

RNase PH is a member of the family of phosphorolytic 3' --> 5' exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X-ray diffraction to medium resolution. Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively. The structures contain a rare left-handed beta alpha beta-motif in the N-terminal portion of the protein. This motif has also been identified in other enzymes involved in RNA metabolism. The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N-terminal core of the structure and active site of Streptomyces antibioticus PNPase. The surface of the RNase PH dimer fit the shape of a tRNA molecule.     

RNase G of Escherichia coli exhibits only limited functional overlap with its essential homologue,RNase E

RNase G (rng) is an E. coli endoribonuclease that is homologous to the catalytic domain of RNase E (rne), an essential protein that is a major participant in tRNA maturation, mRNA decay, rRNA processing and M1 RNA processing. We demonstrate here that whereas RNase G inefficiently participates in the degradation of mRNAs and the processing of 9S rRNA, it is not involved in either tRNA or M1 RNA processing. This conclusion is supported by the fact that inactivation of RNase G alone does not affect 9S rRNA processing and only leads to minor changes in mRNA half-lives. However, in rng rne double mutants mRNA decay and 9S rRNA processing are more defective than in either single mutant. Conversely, increasing RNase G levels in an rne-1 rng::cat double mutant, proportionally increased the extent of 9S rRNA processing and decreased the half-lives of specific mRNAs. In contrast, variations in the amount of RNase G did not alter tRNA processing under any circumstances. Thus, the failure of RNase G to complement rne mutations, even when overproduced at high levels, apparently results from its inability to substitute for RNase E in the maturation of tRNAs.