Organization and Expression of the Polynucleotide Phosphorylase Gene ( pnp ) of Streptomyces : Processing of pnp Transcripts in Streptomyces antibioticus

Abstract

ABSTRACT 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, P pnp , 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 P pnp 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 P pnp 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 P pnp 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.