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Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.