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. 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.
Chang
Samantha A
SA
Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
Bralley
Patricia
P
Jones
George H
GH
D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D009876:Operon; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D006639:Histidine; D011117:Polyribonucleotide Nucleotidyltransferase; D043244:Ribonuclease III