Regulation of Escherichia coli Polynucleotide Phosphorylase by ATP

Abstract

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPasedependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.Polynucleotide phosphorylase (PNPase), 3 a polyribonucleotide nucleotidyltransferase (EC 2.7.7.8), is a homotrimeric enzyme involved in RNA turnover in bacteria and eukaryotic organelles (1). PNPase processively catalyzes the phosphorolysis of RNA in the 3Ј-to 5Ј-direction, thus releasing nucleoside diphosphates, and the reverse 5Ј-to 3Ј-template-independent polymerization of nucleoside diphosphates (2-7). PNPase also binds RNA via its KH and S1 RNA-binding domains located at the C terminus of the polypeptide (6, 8 -11).The monomeric subunit exhibits a five-domain structure that is widely conserved from bacteria to plants and mammals (12, 13). The structural core of the subunit appears to be a duplication of an RNase PH domain, with the two RNase PHlike domains connected by a poorly conserved linker domain. In the doughnut-shaped homotrimeric protein, the subunits form a central channel where catalysis is thought to occur; the KH and S1 RNA-binding domains are located on top of the enzyme, with the former immediately above the central channel and the latter facing outward away from the channel (14).PNPase was originally implicated in the synthesis of cellular RNA before the template-dependent RNA polymerase was discovered (2, 15, 16); later on, because of its phosphorolytic activity, it was implicated in RNA degradation (6). It has long been assumed that, because of the high P i intracellular concentration, PNPase would act in vivo mainly phosphorolytically (17). More recently, however, it was shown that PNPase can add heteropolymeric tails to RNA 3Ј-ends (18,19). Because in Escherichia coli PNPase-dependent heteropolymeric failing, like polyadenylation by polyadenyl polymerase (PAP), targets bacterial RNAs to degradation (20), both PNPase phosphorolytic and polymerization activities participate in PNPase-dependent RNA decay. Finally, it was suggested that PNPase plays a central role in the biosynthetic pathway of dCTP by providing the CDP precursor (21), thus linking RNA turnover to DNA replication.Although widely conserved in Bacteria and Eukarya, the pnp gene does not seem to be essential for s...

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…This results in PNPase activation, while highly aerobic conditions inhibit PNPase (90). Cyclic di-GMP joins a growing group of small molecules that allosterically regulate PNPase activity and RNA turnover (358,359).…”

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq

“…Hfq was also shown to interact with the polynucleotide phosphorylase (PNPase) (Mohanty et al 2004), a major 39-59 exonuclease involved in RNA degradation (Andrade et al 2009b). PNPase responds to environmental stimuli, and its activity is modulated by metabolites such as ATP, citrate, and cyclic di-GMP (Del Favero et al 2008;Nurmohamed et al 2011;Tuckerman et al 2011). We have previously shown that PNPase is a key factor in the turnover of small RNAs controlling the expression of outer membrane proteins in the stationary phase of growth (Andrade and Arraiano 2008).…”

RNA remodeling and gene regulation by cold shock proteins

“…86 The role of CsdA in mRNA degradation was suggested by the observations that it (1) is found in degradosomes in cold-adapted 5'-diphosphate from the 3' end of the substrate and also carries out the reverse reaction of nucleoside 5'-diphosphate polymerization with the release of phosphate; 120 both the phosphorolytic and polymerization activities are allosterically inhibited by ATP. 121 The polymerization activity of PNPase is responsible for residual RNA tailing observed in E. coli mutants devoid of the main polyadenylating enzyme PAP I. 122,123 PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge.…”