The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor

Abstract

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 singlestranded homopolymers. We thus designate the absB product RNase IIIS. Using T7 RNA polymerase and a cloned template containing the rpsO-pnp intergenic region, we synthesized an RNA substrate representing a portion of the read-through transcript normally produced in S. coelicolor. This transcript contains the sequences that form the putative rpsO terminator and those that form an intergenic stem-loop structure thought to be the site for RNase IIIS processing of the read-through transcript. We show that RNase IIIS does cleave that model transcript, with primary and secondary cleavage sites in an internal loop in the stem-loop structure. We have identified the primary and secondary cleavage sites by primer extension and demonstrate the further processing of the initial cleavage products. Thus, as is the case in Escherichia coli, the read-through transcript from rpsO-pnp is cleaved by RNase IIIS in S. coelicolor. However, the cleavage sites are different in the two systems. The positions of the cleavage sites in the stem-loop of the S. coelicolor transcript are more akin to those identified in the processing of bacteriophage T7 mRNAs. A kinetic assay for RNase IIIS was developed, and kinetic parameters for the reaction utilizing the model transcript from rpsO-pnp were determined.Ribonuclease III is a double strand-specific endoribonuclease found in bacteria and eukaryotes. In Escherichia coli, RNase III is involved in the processing and maturation of viral, ribosomal, and messenger RNAs. Studies of the processing of bacteriophage T7 RNAs demonstrated an important role for RNase III and identified sequence features that determine the specificity of cleavage (1-3). RNase III is involved in the processing of the 30 S ribosomal RNA precursor to pre-16 S and pre-23 S intermediates in E. coli, and the 30 S precursor only accumulates in RNase III-deficient mutants (4, 5). Of particular relevance to the present study, RNase III is involved in the processing of transcripts from the rpsO-pnp operon in E. coli. The operon is transcribed from two promoters, PrpsO, situated upstream of rpsO, the gene for ribosomal protein S15, and Ppnp, a promoter located in the intergenic region between rpsO and pnp, the gene for polynucleotide phosphorylase (6). Transcription initiating at PrpsO produces a read-through transcript containing both rpsO and pnp, whereas initiation at Ppnp produces a pnp transcript only (6). Secondary structure models and biochemical studies have confirmed the presence of a stem-loop structure in the rpsO-pnp intergenic region, upstream of the pnp translation start site (6, 7). This stem loop is present in both the read-through transcript and the transcript from Ppnp and is cleaved at identical site...

RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

“…Primers rps1 and ϩ16R are complementary to the readthrough transcript from the rpsO-pnp operon of S. coelicolor, while primers ramR F1 and R1 identified the ramR transcript. SCO5737 (encoding polynucleotide phosphorylase) and SCO3982 to SCO3988 were amplified using primer pairs SCPNPF3 and pnpB1 and SCO3982F1 and R1, respectively (see Table S1), and products were cloned into pCR2.1TOPO as described previously (10). Nonradioactive runoff transcripts from these constructs were prepared using T7 RNA polymerase, as described previously (16).…”

“…Of particular relevance to the present report are the studies of Champness and coworkers (1,2) on the absB locus of S. coelicolor. absB was shown to encode a homolog of E. coli RNase III, and the function of the absB product as a double-strand-specific endoribonuclease has been subsequently verified (1,2,10). Mutations in the absB locus reduce or abolish the production of all four of the antibiotics normally synthesized by S. coelicolor (2).…”

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…Of these, RNase III, a double-strand-specific endonuclease involved in the processing of RNA transcripts (150), plays an important role in the regulation of antibiotic biosynthesis. An S. coelicolor RNase III (absB) mutant was deficient in ACT, RED, MM, and CDA because of low expression of the cognate CSR genes (151).…”

Streptomyces coelicolor Polynucleotide Phosphorylase Can Polymerize Nucleoside Diphosphates under Phosphorolysis Conditions, with Implications for the Degradation of Structured RNAs

“…The first step utilized primer SCPNPF1N (forward) and either SCN459DR2 or SCC468AR2 as the reverse primer (Table 1), with the recombinant plasmid pJSE600 as the template. pJSE600 contains the wild-type S. coelicolor pnp gene (12). These primer sets yielded products of ca.…”

“…We examined the activity of the wild-type and mutant PNPases in kinetic assays for polymerization and phosphorolysis. In previous studies, these in vitro assays were conducted at 37°C (11,12). Because Streptomyces members grow optimally nearer 30°C and because we wished to examine the properties of the enzymes under conditions that were as close as possible to those that might occur in vivo, the kinetic assays in the present study were performed at 30°C.…”