Mono- and dimethylating activities and kinetic studies of the ermC 23 S rRNA methyltransferase

The ermC 23 S rRNA methyltransferase converts a single adenine residue to N6,N6-dimethyladenine, both in vivo and in vitro. The ermC methyltransferase was demonstrated to produce both N6-mono and N6,N6-dimethylated adenine residues in Bacillus subtilis 23 S rRNA during the course of the reaction in vitro. An almost total conversion of monomethylated intermediates into dimethylated products was observed upon completion of the reaction. Data presented here demonstrate that the addition of the two methyl groups to each 23 S rRNA molecule takes place through a monomethylated intermediate and suggest that the enzyme dissociates from its RNA substrate between the two consecutive methylation reactions. The enzyme is able to utilize monomethylated RNA as substrate for the addition of a second methyl group with an efficiency approximately comparable to that obtained when unmethylated RNA was the initial substrate. Initial-rate data and inhibition studies suggest that the ermC methylase reaction involves a sequential mechanism occurring by two consecutive Random Bi Bi reactions.     

Substrate requirements for ErmC' methyltransferase activity.

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.     

Effect of polyamines on in vitro reconstitution of ribosomal subunits

The effect of polyamines on in vitro reconstitution of Escherichia coli 30S and 50S ribosomal subunits has been studied. Spermidine stimulated the reconstitution of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits at least 1.6-fold. The reconstitution of 30S particles from normal 16S rRNA and total proteins of 30S subunits exhibited only slight spermidine stimulation. However, the optimal Mg2+ concentration of the reconstitution was decreased from 20 mM to 16 mM in the presence of 3 mM spermidine. In the absence of spermidine the assembly of 30S particles from normal 16S rRNA was more rapid than the assembly from 16S rRNA lacking the methyl groups on two neighboring adenines. The reconstitution of 50S particles from 23S and 5S rRNA and total proteins of 50S subunits was not influenced greatly by spermidine. Gel electrophoresis results, from reconstitution experiments of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits, showed that the assembly of S1 and S9 proteins to 23S core particles was stimulated by spermidine during reconstitution. The relationship of polyamine effects on in vitro ribosome assembly from its constituents to in vivo ribosome assembly is discussed. The reconstitution of Bacillus subtilis 30S particles from 16S rRNA and total proteins of 30S subunits was also stimulated approximately 1.3-fold by 3 mM spermidine.     

General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation.

A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper.     

Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA.

Four previously undetermined sites of methylation are mapped in Escherichia coli 23S rRNA employing a novel combination of methods. First, using a double-isotope approach, the total number of methyl groups in 23S rRNA was determined to be 14.9 +/- 1.6. Second, hybridization of methyl-labeled rRNA to complementary DNA restriction fragments and PAGE analysis were used to purify RNA-DNA heteroduplexes and to quantify methyl groups within specific 23S rRNA fragments. Third, the methylated nucleosides in these fragments were identified and quantified using HPLC, confirming the presence of 14 methylation sites in 23S rRNA, four more than had been previously identified. In contrast, a similar set of analyses conducted on 16S rRNA gave evidence for 10 sites of methylation, at all approximate locations consistent with published 16S methylated nucleoside identities and locations. Selected regions of the 23S rRNA molecule containing previously unidentified methylated nucleosides were released by site-directed cleavage with ribonuclease H and isolated by PAGE. Sites of methylation within the RNA fragments were determined by classical oligonucleotide analyses. The four newly identified methylation sites in 23S rRNA are m2G-1835, m5C-1962, m6A-2503, and m2G at one of positions 2445-2447. Together with previously described sites of modification, these new sites form a group that is clustered in a current model for the three-dimensional organization of the 23S rRNA in the 50S ribosomal subunit, at a locus congruent with nucleotides previously implicated in ribosomal function.     

Characterization of a plasmid-specified ribosome methylase associated with macrolide resistance.

The ermC gene of plasmid pE194 specifies resistance to the macrolidelincosamide-streptogramin B antibiotics. This resistance, as well as synthesis of the 29,000 dalton protein product of ermC, has been shown to be induced by erythromycin. Weisblum and his colleagues have established that macrolide resistance is associated with a specific dimethylation of adenine in 23 S rRNA. We show that pE194 specifies an RNA methylase that can utilize either 50 S ribosomes or 23 S rRNA as substrates. Synthesis of this methylase is induced by low concentrations of erythromycin, and the enzyme is produced in elevated amounts by strains carrying a high copy number mutant of pE194. The methylase comigrates with the 29K ermC product on polyacrylamide gels. The purification and some properties of this methylase are described.     

Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.     

Ribosomal RNA methylation in Staphylococcus aureus and Escherichia coli: effect of the "MLS" (erythromycin resistance) methylase

Classical acquired resistance to erythromycin in Staphylococcus aureus ("MLS," or macrolide-lincosamide-streptogramin, resistance) was shown by Weisblum and colleagues to be a direct consequence of the conversion of one or more adenosine residues of 23S rRNA, within the subsequence(s) GA3G, to N6-dimethyladenosine (m62A). The methylation reaction is effected by a class of methylase, whose genes are typically plasmid- or transposon-associated, and whose synthesis is inducible by erythromycin. Using a recently obtained clinical MLS isolate of S. aureus, we have further defined the methylation locus as YGG X m62A X AAGAC; and have shown that this subsequence occurs once in the 23S RNA and that it is essentially completely methylated in all copies of 23S RNA that accumulate in induced cultures. Similar findings were obtained with laboratory S. aureus strains containing two well-characterized evolutionary variants (ermB, ermC) of MLS methylase genes. Analyses of a strain of E. coli containing the ermC gene indicated that the specificity of the methylase gene was unchanged, but that its expression was muted. Even after prolonged periods of induction, the strain manifested only partial resistance to erythromycin, and only about one-third of the copies of the MLS subsequence were methylated in such "induced" cultures. Since the E. coli 23S RNA sequence is known in its entirety, localization of the MLS subsequence is in this case unambiguous; as inferred by homology arguments applied earlier to the S. aureus data, the subsequence is in a highly conserved region of 23S RNA considered to contribute to the peptidyl transferase center of the ribosome.     

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.     

Ribosomal ribonucleic acid synthesis in Bacillus subtilis

The mode of biosynthesis of the 16S and 23S ribosomal ribonucleic acids (rRNA) was studied in Bacillus subtilis 168thy(-). Three criteria were used to define the characteristics of the rRNA species: (i) the time required at 37 degrees C to synthesize 16S and 23S rRNA chains de novo in growing cultures; (ii) the degree of reactivity of the 3'-terminal groups of the rRNA molecules with periodate and [carbonyl-(14)C]isonicotinic acid hydrazide; and (iii) the reactivity of the 5'-terminal regions of the rRNA molecules with the bacterial exonuclease purified by Riley (1969). The 16S and 23S chains of B. subtilis were synthesized at rates of 22+/-2 and 21+/-2 nucleotides added/s. The periodate-[(14)C]isonicotinic acid hydrazide and the exonuclease techniques for estimating apparent chain lengths of RNA indicated that the chain length of the 23S rRNA was 1.8 times that of the 16S fraction. The apparent chain lengths of each rRNA species were: 16S rRNA, 1650+/-50 nucleotide residues; 23S rRNA, 3050+/-90 nucleotide residues. It appears that, the 16S and 23S rRNA molecules in B. subtilis are synthesized in the expected manner, by simple polymerization of the final products on independent cistrons.     

The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase

Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ.     

Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes.

The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.     

Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

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.     

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.     

Isolation and characterization of a nucleolar 2'-O-methyltransferase from Ehrlich ascites tumor cells

A 2'-O-methyltransferase that transfers the methyl group from S-adenosylmethionine to the 2'-hydroxyl group of ribose moieties of RNA has been purified from Ehrlich ascites tumor cell nucleoli. The partially purified enzyme is devoid of other RNA methylase activities and is free of ribonucleases. The enzyme has optimal activity in tris(hydroxymethyl)aminomethane buffer, pH 8.0, in the presence of 0.4 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, and 50 mM KCl, and has an apparent Km for S-adenosylmethionine of 0.44 microM. Gel filtration studies of this enzyme gave a Stokes radius of 43 A. Sedimentation velocity measurements in glycerol gradients yield an S20,w of 8.0 S. From these values, a native molecular weight of 145,000 was calculated. The enzyme catalyzes the methylation of synthetic homoribopolymers as well as 18S and 28S rRNA; however, poly(C) is the preferred synthetic substrate, and preference for unmethylated sequences of rRNA was observed. For each RNA substrate examined, only methylation of the 2'-hydroxyl group of the ribose moieties was detected.