# Novel Aspects of Polynucleotide Phosphorylase Function in _Streptomyces_
## 3 PNPase Function as an RNA 3\({}^{\prime}\)-Polyribonucleotide Polymerase in _Streptomyces_  
As is the case in eukaryotes, bacterial RNAs have oligo- and polyribonucleotide tails at their 3\({}^{\prime}\)-ends, and these tails are added post-transcriptionally (reviewed in [37]). In _E. coli_, the primary enzyme responsible for the synthesis of these tails is poly(A) polymerase I (PAP I), and the tails are composed primarily of A residues [38]. In bacteria, poly(A) tails function to facilitate RNA degradation as the major 3\({}^{\prime}\)-exoribonucleases, viz. RNase II and PNPase in _E. coli_, digest these tails processively in vitro and in vivo [8; 39]. Figure 3: Cold shock responses of _S. coelicolor_. Derivatives containing the _rpsO–pnp_ promoter probe constructs were grown and 30 \({}^{\circ}\)C, and half of each culture was then shifted to 10 \({}^{\circ}\)C. Mycelium was harvested at the indicated times, disrupted by sonication, and following centrifugation, supernatants were assayed for promoter activity, as described [31; 35]. Panel **C** shows the results of PNPase polymerization assays. In Panels **A** and **B**, PNPase promoter activities are expressed relative to the activity measured at 30 \({}^{\circ}\)C at zero time, immediately before the shift to 10 \({}^{\circ}\)C. The results shown are the averages of duplicate assays from two independent experiments \(\pm\) SEM. In the first experiment, PNPase levels were measured in _S. coelicolor_ containing P_rpsO_A_ and in the second, PNPase levels were measured in the derivative containing P_pnp_B. This figure is reprinted from [PERSON], 536, [PERSON], [PERSON], [PERSON], Transcription of the _rpsO-pnp_ operon of _Streptomyces coelicolor_ involves four temporally regulated, stress responsive promoters. 177–185, Copyright (2014), with permission from Elsevier [31]. It was shown some years ago by [PERSON] and [PERSON] that an _E. coli_ mutant lacking PAP I still added tails to the 3\({}^{\prime}\)-ends of its RNAs. However, these tails were not composed exclusively of A residues; the tails contained G, C, and U residues as well, i.e., they were heteropolymeric [40]. [PERSON] and [PERSON] demonstrated that the enzyme responsible for the synthesis of these heteropolymeric tails was none other than PNPase [40]. Thus, even in the absence of PAP I, PNPase can add 3\({}^{\prime}\)-tails to facilitate the degradation of cellular RNAs. _Streptomyces_ do not contain PAP I. Yet the 3\({}^{\prime}\)-ends of streptomycincte RNAs do possess tails. Moreover, the tails are heteropolymeric in composition, like those synthesized by PNPase in _E. coli_ in the absence of PAP I [41; 42]. These observations, and the report that PNPase functions as the RNA 3\({}^{\prime}\)-polyribonucleotide polymerase in plant chloroplasts and in cyanobacteria [43; 44], led to the hypothesis that PNPase played the same role in _Streptomyces_[45]. The straightforward way to test this hypothesis would be to create a _pnp_ null mutant, e.g., in _S. coelicolor_, and to determine whether the mutant was still capable of adding 3\({}^{\prime}\)-tails to its RNAs. However, attempts to disrupt _pnp_ in _S. coelicolor_ and in the sister species, _Streptomyces antibioticus_, were only successful when a second copy of _pnp_ was added to the genome. In other words, gene disruption attempts revealed that, unlike the situation in _E. coli_ and in the Bacilli, _pnp_ is an essential gene in _Streptomyces_[42]. A model for the function of PNPase as an RNA 3\({}^{\prime}\)-polyribonucleotide polymerase is presented in detail below. Function of PNPase as Both an Exoribonuclease and as an RNA 3\({}^{\prime}\)-Polyribonucleotide Polymerase  
PNPase activity is highly processive and the enzyme is impeded by stem-loop structures [46]. Streptomycescte genomes are GC rich, so that enzymes involved in RNA decay may have evolved to degrade the RNAs derived from these genomes efficiently.