Involvement of the mature domain in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

A precursor of 5S ribosomal RNA from Bacillus subtilis (p5A rRNA, 179 nucleotides in length) is cleaved by RNase M5, a specific maturation endonuclease which releases the mature 5S rRNA (m5, 116 nucleotides) and precursor fragments derived from the 5' (21 nucleotides) and 3' (42 nucleotides) termini of p5A rRNA. Previous results (Meyhack, B., et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3045) led to the conclusion that recognition elements in potential RNase M5 substrates mainly reside in the mature moiety of the precursor. Limited digestion of p5A rRNA with RNase T1 permitted the isolation of a number of test substrates which contained both precursor-specific segments and were unaltered in the immediate vicinity of the cleavage sites, but which differed in that more or less extensive regions of the mature moiety of the p5A rRNA were deleted. Tests of the capacity of these partial molecules to serve as substrates for RNase M5 indicate clearly that the enzyme recognizes the overall conformation of potential substrates, neglecting only the double-helical "prokaryotic loop" (Fox, G.E., & Woese, C.R. (1975) Nature (London) 256, 505).     

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.     

Involvement of precursor-specific segments in the in vitro maturation of Bacillus subtilis precursor 5S ribosomal RNA.

In vitro maturation of precursor 5S ribosomal RNA (p5A) from Bacillus subtilis effected by RNase M5 yields mature 5S RNA (m5, 116 nucleotides), and 3' precursor-specific segment (42 nucleotides), and a 5' precursor-specific segment (21 nucleotides) (Sogin, M.L., Pace, B., and Pace, N.R. (1977), J. Biol. Chem. 252, 1350). Limited digestion of p5A with RNase T2 introduces a single scission at position 60 of the molecule; m5 is cleaved at the corresponding nucleotide residue. The complementary "halves" of the molecules could be isolated from denaturing polyacrylamide gels. The isolated fragments of p5A are not substrates for RNase M5, suggesting that some recognition elements can be utilized by RNase M5 only when presented in double-helical form. In exploring the involvement of the precursor-specific segments in the RNase M5-p5A interaction, substrate molecules lacking the 3' or 5' precursor-specific segment were constructed by reannealing complementary "halves" from p5A and m5 RNA. The artificial substrate lacking the 5'-terminal precursor segment was cleaved very much more slowly than the lacking t' segment; the 5' precursor-specific segment therefore contains one or more components recognized by RNase M5 during its interaction with the p5A substrate.     

The ribonucleoprotein substrate for a ribosomal RNA-processing nuclease

The Bacillus subtilis RNase M5 activity, responsible for the endonucleolytic maturation of 5 S rRNA, requires two proteins, alpha and beta. The beta component has been purified to homogeneity and shown to correspond to ribosomal protein BL16. The BL16 protein evidently corresponds functionally to Escherichia coli ribosomal protein EL18, as that latter protein also will complement the B. subtilis alpha protein in the RNase M5 reaction. A filter binding assay for the formation of B. subtilis 5 S rRNA-protein complexes was characterized and used to evaluate the association of BL16 protein with some RNAs. A native precursor of 5 S rRNA, containing extra sequences at both termini of the mature domain, binds the ribosomal protein no better than the mature 5 S rRNA; the precursor sequences do not facilitate that interaction. A model is considered in which the precursor segments facilitate, by refolding, the dissociation of processing products prior to the RNase M5 step. Electrostatic versus nonelectrostatic contributions to the BL16-5 S rRNA complex formation were inspected by analyzing variation in apparent association constants as a function of ionic strength. Electrostatic interactions were seen to contribute approximately 65% to the overall binding energy.     

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.     

Partial purification and properties of a ribosomal RNA maturation endonuclease from Bacillus subtilis.

Data are presented on the partial purification and properties of a 5 S ribosomal RNA maturation nuclease, termed RNase M5, from Bacillus subtillis 168. RNase M5 specifically cleaves 21 and 42 nucleotides, respectively, from the 5' and 3' termini of a 5 S rRNA precursor to yield the mature (116 nucleotides) 5 S rRNA. The cleavage is endonucleolytic with the formation of 5'-phosphoryl and 3'-hydroxyl groups. Enzyme action requires divalent cations, which may be furnished by either certain metals or by polyamines. The activity is separable into two components both of which are required for activity. It appears that the same nuclease excises the 5'- and 3'-terminal segments since preparations lose the capacity to modify the two termini with an identical first order thermal decay rate. Certain features of the rRNA precursor which may be involved in cognitive interaction with RNase M5 are discussed.     

Recognition signals for mouse pre-rRNA processing

In order to identify signals for rRNA processing in eukaryotes, mouse pre-rRNA sequence features around four cleavage sites have been analyzed. No consensus sequence can be recognized when the four boundary regions are examined. Unlike mature rRNA termini, distal sequences of precursor-specific domains cannot participate in stable duplex with adjacent regions. The extensive divergence of precursor-specific sequences during evolution also applies to nucleotides adjacent to cleavage sites, with a significant exception for a conserved segment immediately downstream 5.8S rRNA. A specific role is proposed for U3 nucleolar RNA in the conversion of 32S pre-rRNA into mature 28S rRNA, through base-pairing with precursor-specific sequences at the boundaries of excised domains.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli 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.     

The catalytic element of a ribosomal RNA-processing complex

The Bacillus subtilis RNase M5 complex, responsible for the terminal maturation of 5 S rRNA, includes two proteins. One of these proteins is ribosomal protein BL16 (equivalent to Escherichia coli EL18); the other, the alpha component, is required for catalysis. The RNase M5 alpha component has been purified in bulk extensively, and the active polypeptide (Mr approximately 24,000) identified following polyacrylamide gel electrophoresis. Reaction conditions (20-30% dimethyl sulfoxide) are reported which render RNase M5 activity independent of ribosomal protein BL16. This proves that alpha indeed is the catalytic element, the actual RNase M5, which normally attacks a ribonucleoprotein substrate consisting of protein BL16 in complex with the 5 S rRNA precursor. Kinetic analyses of the BL16-dependent and independent reactions suggest that any alpha-BL16 association contributes little to the energetics of the alpha-ribonucleoprotein substrate interaction. It is postulated that the BL16 protein serves as a scaffold, to lock the precursor mRNA into a conformation recognizable by the nuclease.     

Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.     

Escherichia coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing of the 5' end of 16S rRNA.

We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA. This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5' end processing of 16S rRNA. Accumulation of 16. 3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene. The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region. A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant. This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant. These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5' end of 16S rRNA.     

Some characteristics of processing sites in ribosomal precursor RNA of yeast.

The DNA sequences of the intergenic region between the 17S and 5.8S rRNA genes of the ribosomal RNA operon in yeast has been determined. In this region the 37S ribosomal precursor RNA is specifically cleaved at a number of sites in the course of the maturation process. The exact position of these processing sites has been established by sequence analysis of the terminal fragments of the respective RNA species. There appears to be no significant complementarity between the sequences surrounding the two termini of the 18S secondary precursor RNA nor between those surrounding the two termini of 17S mature rRNA. This finding implies that the processing of yeast 37S ribosomal precursor RNA is not directed by a double-strand specific ribonuclease previously shown to be involved in the processing of E. coli ribosomal precursor RNA [see Refs 1,2]. The processing sites of yeast ribosomal precursor RNA described in the present paper are all flanked at one side by a very [A+T]-rich sequence. In addition, sequence repeats are found around the processing sites in this precursor RNA. Finally, sequence homologies are present at the 3'-termini [6 nucleotides] and the 5'-termini [13 nucleotides] of a number of mature rRNA products and intermediate ribosomal RNA precursors. These structural features are discussed in terms of possible recognition sites for the processing enzymes.