# Novel Aspects of Polynucleotide Phosphorylase Function in _Streptomyces_
## 3 PNPase Function as an RNA 3\({}^{\prime}\)-Polyribonucleotide Polymerase in _Streptomyces_
A possible strategy for facilitating this degradation was suggested by the observation that PNPase appears to utilize its polymerizing activity to add 3\({}^{\prime}\)-tails to streptomyccte RNAs [42; 45]. It seemed possible that the enzyme might add such tails during phosphorylosis to create single stranded 3\({}^{\prime}\)-ends that would then function as the substrates for that phosphorylosis. If this were the case, it might be expected that nucleoside diphosphates, the substrates for polymerization, would stimulate phosphorylosis. To test this hypothesis, two model PNPase substrates were constructed from the sequence of the _rpsO-pnp_ operon of _S. coelicolor_. Both substrates contained the _rpsO-pnp_ terminator and the intergenic hairpin. Thus, both model substrates contained secondary structure that would be expected to impede phosphorylosis by PNPase. One substrate, designated 5601, also possessed a single stranded 3\({}^{\prime}\)-tail, 33 bases in length, while the other substrate, 5650, terminated at the base of the intergenic hairpin, and did not have a single stranded tail [47]. The phosphorylosis of these two substrates was studied in the absence and presence of a mixture of all four nucleoside diphosphates (NDPs) with the interesting result, predicted by our hypothesis, that the NDPs, normally the substrates for the polymerizing activity of PNPase, did stimulate RNA degradation by phosphorylosis. Figure 4 shows the results of phosphorylosis of the 5650 transcript (labeled RP3 in the figure). Analysis of the results shown in Figure 4 revealed that NDPs at 20-30 uM in phosphorylosis mixtures stimulated that reaction by 2-3 fold as compared with controls, but only when the structured RNA, 5650 (RP3) was used as the substrate [47]. NDPs had no effect on the phosphorylosis of the 5601 substrate, possessing the single stranded tail. Kinetic analyses showed that NDPs affected the \(K_{\rm m}\) for phosphorylosis. Thus, the \(K_{\rm m}\) value for phosphorylosis of the 5650 substrate in the absence of NDPs was 3.1 uM. This value decreased to 0.65 uM in the presence of all four NDPs at 20 uM. This latter \(K_{\rm m}\) was almost identical to that obtained in the absence of NDPs for the 5601 substrate, which has a single stranded 3\({}^{\prime}\)-tail (0.62 uM). NDPs did not further decrease the \(K_{\rm m}\) for the 5601 substrate. It is noteworthy as well, that NDPs had no effect on the phosphorylosis of either substrate by _E. coli_PNPase, and that the _E. coli_ enzyme was intrinsically less active with the structured substrate than was its counterpart from _S. coelicolor_[47]. Our model for the explanation of the foregoing observations is shown in Figure 5[49]. We posit that the stem-loops of structured substrates, like 5650, block PNPase action. The addition of short 3\({}^{\prime}\)-tails during phosphorolysis, or the presence of naturally occurring tails on substrates like 5601, allows for the breathing of stems and thus permits PNPase, which might otherwise stall at the stem-loops in structured substrates, to continue phosphorolysis through those structures. As indicated above, this mechanism may represent an evolutionary adaptation occasioned by the high GC content of streptomycete genomes and their transcripts. It must be noted, however, that the evidence for the function of PNPase as a 3\({}^{\prime}\)-polyribonucleotide polymerase in _Streptomyces_ is indirect. In vivo evidence for this function remains to be uncovered. Figure 4: Effects of nucleoside diphosphates on the phosphorolysis of the 5650 transcript. Phosphorolysis reactions were performed as described in [47], and reaction products were separated by gel electrophoresis. The top panel shows the results obtained with _S. coelicolor_ PNPase and the bottom panel results using _E. coli_ PNPase. Reactions were conducted in the presence of increasing concentrations of a mixture of ADP, CDP, UDP, and GDP (nucleoside diphosphates (NDPs)) as indicated.