# Novel Aspects of Polynucleotide Phosphorylase Function in _Streptomyces_
## 6 Conclusions and Unanswered Questions  
It is apparent from the brief analysis above that PNPase is a multitalented enzyme that plays a critical role in the metabolic activities of bacterial cells. Despite the wealth of information that has been accumulated about PNPase, a number of important biological questions remain unanswered, particularly as they relate to the functions of _Streptomyces_ PNPase.  
First, why is the _rpsO-pnp_ operon of _S. coelicolor_ transcribed from four promoters? The answer to this question may relate to the fact that the operon contains a ribosomal protein gene, as well as the gene for PNPase. It is possible that the promoters are not only critical to the regulation of _pnp_ expression, but that they also play and important role in ribosome biogenesis via their regulation of the levels of the _rpsO_ transcript. Mutation of the four promoter sequences may provide insight into these possibilities.  
Second, what is the significance, if there is any, to the fact that the _S. coelicolor rpsO-pnp_ operon produces six transcripts (two _rpsO_ transcripts from P_rps_OA and B, two readthrough transcripts from the same two promoters, and two _pnp_ transcripts from P_pnp_A and B)? It should be noted that Northern blot analysis of the transcripts derived from the _S. coelicolor rpsO-pnp_ operon did not reveal the presence of six separate transcripts [65]. It is possible that the different transcripts are not sufficiently different in size to have been resolved on the Northern blotting gels. Another intriguing possibility is that the longer transcripts, obtained from the upstream promoter in each case, might be processed at their 5\({}^{\prime}\)-ends by RNase J, which possesses 5\({}^{\prime}\)-3\({}^{\prime}\)-exoribonuclease activity. RNase J has recently been characterized in _S. coelicolor_[66; 67].  
Third, as described above, PNPase is a cold shock protein in _Streptomyces_. It is relevant to ask whether PNPase responds to other environmental stresses, such as heat, oxidative stress, metal ion stress, etc. It would be of interest, in particular, to examine the effects of various types of stress on the activities of the _rpsO-pnp_ promoters. Mutational analyses again might reveal important aspects of promoter function in stress conditions in _Streptomyces_.  
Fourth, a number of small effector molecules modulate the activity of _E. coli_ PNPase, e.g., ATP, citrate, and cyclic-diGMP [16; 17; 18; 19]. It has been proposed that these effectors connect RNA decay to other metabolic pathways in bacterial cells. It would be interesting to determine whether these effectors also affect the activity of _Streptomyces_ PNPase. It is noteworthy, in this regard, that in silico molecular docking studies suggest that citrate, which inhibits the activity of _E. coli_ PNPase, will bind to PNPase from _S. antibioticus_[18].  
Finally, in _E. coli_ and other organisms, PNPase is part of a larger macromolecular complex generally referred to as the degradosome ([68; 69]. In _E. coli_, the components of the degradosome are organized around a scaffold provided by the single strand specific endoribonuclease, RNase E [70]. RNase E is present in _S. coelicolor_, and has been shown to interact with PNPase in vivo [71]. However, unlike the situation in _E. coli_, the identities of other proteins that might be involved in the degradative machine are unknown in _Streptomyces_.  
It is fervently hoped that the foregoing and other important questions related to PNPase structure and function will continue to attract interest and experimentation to provide answers to them.  
Much of the research described in this report was supported by grants MCB-0133520 and MCB-0817177 from the U.S. National Science Foundation to the author.  
The author declares no conflicts of interest.