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. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.
Bralley
Patricia
P
Department of Biology, Emory University, Atlanta, Georgia 30322, USA. pbralley@biology.emory.edu
Jones
George H
GH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003198:Computer Simulation; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013303:Streptomyces antibioticus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D011117:Polyribonucleotide Nucleotidyltransferase