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 single-stranded 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.
Highlights
Containing both rpsO and pnp, whereas initiation at Ppnp produces a pnp transcript only [6]
Those cleavage sites occur in a different position in the stem-loop as compared with the sites in the rpsO-pnp transcripts in E. coli, and these results coupled with our earlier observations [10] suggest that the processing of rpsO-pnp transcripts in Streptomyces differs in important respects from the corresponding process in E. coli
Complementation of the absB mutation followed by DNA sequencing identified an open reading frame with significant homology to bacterial RNase III [12]
Summary
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 sites in the two transcripts by RNase III [6, 7]. Unlike the situation in E. coli, only the read-through transcript in Streptomyces is processed by RNase III cleavage, whereas the transcript from Ppnp may be processed by a single strand-specific endonuclease like RNase E [10]. Our in vitro data are consistent with those obtained in vivo [10] and summarized above for the processing of the rpsO-pnp intergenic region in S. antibioticus
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