# Novel Aspects of Polynucleotide Phosphorylase Function in _Streptomyces_
## 2 Organization and Expression of the PNPase Gene in _Streptomyces_  
In _E. coli_, and other organisms that have been studied, the PNPase gene, _pnp_, is a part of an operon that also includes _rpsO_, the gene for ribosomal protein S15 [9; 25; 26]. That operon is transcribed from two promoters in _E. coli_, designated _PrpsO_ and _Pnp_[25; 27]. _PrpsO_ is situated upstream of the _rpsO_ gene, and _Pnp_ is located in the intergenic region between the two genes. Transcription from _PrpsO_ ends at a rho-independent terminator and produces the _rpsO_ transcript, but transcription through this terminator occurs with significant frequency and produces a readthrough transcript, containing both _rpsO_ and _pnp_. Transcription from the intergenic promoter, _Pnp_, produces a transcript containing _pnp_ only [25; 27]. In addition to the _rpsO_ terminator, the _rpsO-pnp_ intergenic region contains a second stem-loop that functions as a processing site for the double strand specific endoribonuclease, RNase III. RNase III processing plays an important role in _pnp_ expression [28; 29; 30; 25]. Our interest in the mechanisms of RNA decay in _Streptomyces_ led to an examination of the transcriptional organization of the _rpsO-pnp_ operon in _Streptomyces coelicolor_, the paradigm for biological studies in the genus. To our surprise, primer extension analysis of RNAs isolated from a parental strain of _S. coelicolor_ and from an RNase III null mutant revealed not two, but four extension products, suggesting the presence of four promoters within the _rpsO-pnp_ operon [31]. A visual inspection of 162 putative _Streptomyces_ promoters from [PERSON] [32] and [PERSON] et al. [33], and a group of synthetic promoters generated by [PERSON] et al. [34], indicated that all four of the 5\({}^{\prime}\)-ends identified by primer extension in our studies were preceded by sequences similar to the \(-10\) and \(-35\) regions of characterized streptomycete promoters. Promoter probe cloning of DNA fragments containing these putative promoter sequences verified that the _rpsO-pnp_ operon of _S. coelicolor_ is transcribed from four promoters, two situated upstream of _rpsO_ (PrpsOA and _PrpsOB_) and two situated in the intergenic region, upstream of _pnp_ (P_pnp_A and _Pnp_B, Figure 1). Of particular interest in this analysis was the observation that the four promoters were temporally regulated. That is, the activity of the four promoters varied with time after inoculation of liquid cultures and the variations were promoter-specific, as shown in Figure 2. In terms of maximal activity, _Pnp_B was most active followed by _Prps_OB, _P  
Figure 1: Schematic representation of the _Streptomyces coelicolor rpsO–pnp_ operon. _Prps_OA, B and _Pnp_A, B represent the upstream and intergenic promoters found in _S. coelicolor_, respectively. The ball-and-stick structures immediately following _rpsO_ and _pnp_ represent rho-independent transcription terminators. The ball-and-stick structure just upstream of _pnp_ represents the intergenic hairpin which is cleaved by RNase III. The diagram is not drawn to scale. A major mechanism for the modulation of promoter activity in bacterial systems involves the use of alternative sigma factors by RNA polymerase. The _S. coelicolor_ genome encodes over sixty alternative sigma factors, many of which play roles in differentiation and responses to stress (reviewed in [36]). It was of considerable interest to determine whether the four _rpsO-pnp_ promoters might require alternative sigma factors for transcription in _S. coelicolor_. To this end, we obtained null mutants for a number of sigma factors, including \(\sigma^{\text{B}}\), \(\sigma^{\text{H}}\), and \(\sigma^{\text{L}}\)[36], and their corresponding parental strains. We transferred the _rpsO-pnp_ promoter probe constructs to each strain and measured promoter activity. The results obtained for the \(\sigma^{\text{H}}\) and \(\sigma^{\text{L}}\) mutants were quite similar to those for the parental strain of _S.