Linkage of catalysis and 5' end recognition in ribonuclease RNase J.

In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-β-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.

SCO5745, a bifunctional RNase J ortholog, affects antibiotic production in Streptomyces coelicolor.

The bacterial RNases J are considered bifunctional RNases possessing both endo- and exonucleolytic activities. We have isolated an RNase J ortholog from Streptomyces coelicolor encoded by the gene sco5745. We overexpressed a decahistidine-tagged version of SCO5745 and purified the overexpressed protein by immobilized metal ion affinity chromatography.

Transcription of the rpsO-pnp operon of Streptomyces coelicolor involves four temporally regulated, stress responsive promoters.

Primer extension with RNA from an RNase III null mutant of Streptomyces coelicolor M145 and a primer complementary to the polynucleotide phosphorylase gene revealed two major extension products. Two different extension products were observed using RNA from either wild type M145 or the null mutant with a primer complementary to rpsO. Mapping of the 5'-ends of these extension products to the rpsO-pnp intergenic region indicated that all four putative transcription start sites were preceded by possible promoter sequences.

RNA-Seq and RNA immunoprecipitation analyses of the transcriptome of Streptomyces coelicolor identify substrates for RNase III.

RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain.

RNase III-dependent expression of the rpsO-pnp operon of Streptomyces coelicolor.

We have examined the expression of the rpsO-pnp operon in an RNase III (rnc) mutant of Streptomyces coelicolor. Western blotting demonstrated that polynucleotide phosphorylase (PNPase) levels increased in the rnc mutant, JSE1880, compared with the parental strain, M145, and this observation was confirmed by polymerization assays. It was observed that rpsO-pnp mRNA levels increased in the rnc mutant by 1.

Geobacter sulfurreducens contains separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.

The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed.

RNA 3'-tail synthesis in Streptomyces: in vitro and in vivo activities of RNase PH, the SCO3896 gene product and polynucleotide phosphorylase.

As in other bacteria, 3'-tails are added post-transcriptionally to Streptomyces coelicolor RNA. These tails are heteropolymeric, and although there are several candidates, the enzyme responsible for their synthesis has not been definitively identified. This paper reports on three candidates for this role.

A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases.

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily.

The absB gene encodes a double strand-specific endoribonuclease that cleaves the read-through transcript of the rpsO-pnp operon in Streptomyces coelicolor.

The absB locus of Streptomyces coelicolor encodes a homolog of bacterial RNase III. We cloned and overexpressed the absB gene product and purified a decahistidine-tagged version of the protein. We show here that AbsB is active against double-stranded RNA transcripts derived from synthetic DNAs but is inactive with single-stranded homopolymers.

Addition of poly(A) and heteropolymeric 3' ends in Bacillus subtilis wild-type and polynucleotide phosphorylase-deficient strains.

Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli, poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3' tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3'-tail addition in Bacillus subtilis, which lacks an identifiable PAP I homologue, was studied.

Organization and expression of the polynucleotide phosphorylase gene (pnp) of Streptomyces: Processing of pnp transcripts in Streptomyces antibioticus.

We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S.

Overexpression of the polynucleotide phosphorylase gene (pnp) of Streptomyces antibioticus affects mRNA stability and poly(A) tail length but not ppGpp levels.

The pnp gene, encoding the enzyme polynucleotide phosphorylase (PNPase), was overexpressed in the actinomycin producer Streptomyces antibioticus. Integration of pIJ8600, bearing the thiostrepton-inducible tipA promoter, and its derivatives containing pnp into the S. antibioticus chromosome dramatically increased the growth rate of the resulting strains as compared with the parent strain.

cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor.

In Escherichia coli the poly(A) tails of messenger and rRNAs are a major determinant of RNA stability. These tails are formed primarily by poly(A) polymerase I (PAP I) in wild-type strains or by polynucleotide phosphorylase (PNPase) in PAP I-deficient strains. In Streptomyces coelicolor it has been shown that mycelial RNAs display biochemical characteristics consistent with the presence of poly(A) tails.