S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation

Abstract

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ␤-galactosidase levels reflecting autoregulation and apparent K D values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. Polynucleotide phosphorylase (PNPase) is a conserved, widely distributed phosphorolytic 3=-5= exoribonuclease that may also function under some circumstances as a template-independent RNA polymerase (1; reviewed in reference 2). Although it is not essential, deletions of its gene (pnp) are synthetically lethal when either RNase II or RNase R, both of which are hydrolytic 3=-5= exoribonucleases, is deficient (3-5). Strains deficient in PNPase are also sensitive to cold shock and other stresses (6-9). Thus, PNPase is believed to play significant roles in mRNA turnover and other aspects of RNA processing and metabolism (4, 10-12). Partial or full structures of PNPase from several microorganisms (13-16), as well as structures of the related archaeal exosome (17, 18), have shed considerable light on its mechanism of action. Bacterial PNPase is composed of three identical subunits. Each subunit consists of two tandem globular domains (residues 8 to 210 and 312 to 541; see Fig. 1a) derived from RNase PH that form a core whose central channel is accessible from both the upper and lower surfaces of the core (14,19). Each subunit also contains a C-terminal extension that consists of two additional small domains, KH and S1 (residues 551 to 591 and 622 to 691, respectively; Fig. 1), which are positioned on the upper surface of the core. The KH and S1 domains have been implicated in autoregulation (20), in resistance to cold shock (7), in pathogenesis (21), and in substrate binding (22). Although a solution structure of the S1 domain from Escherichia coli PNPase has been available (13), the positions of the S1 and KH domains relative to the core have been elucidated only recently from the structure of PNPase from Caulobacter crescentus (16). The S1 and KH domains do not appear to be required for the enzymatic activity of PNPase (20,22,23). Nonetheless, deletion of the S1, the KH or both domains results in significant loss of RNA binding and inefficient enzymatic turnover (22).PNPase from E. coli and other organisms exhibits strong autoregulation...

Direct observation of processive exoribonuclease motion using optical tweezers

“…PNPase mutants in which the KH and S1 RNA-binding domains were deleted have been reported to display reduced nucleolytic activity (17,40). We considered whether removal of these domains had any effect on velocity or processivity.…”

“…Each protomer also carries a KH and an S1 RNA-binding domain at its C terminus, both of which are connected to the body of the enzyme by flexible linkers (6, 16). These domains are thought to bind RNA upstream of the catalytic sites, and recent structural evidence suggests that they may guide the substrate into the central channel through a "hands gripping a rope" mechanism (16,17). By itself, PNPase may stall on structured RNA, particularly on G:C hairpins comprising more than six base pairs (18).…”

KH–RNA interactions: back in the groove

“…Specific recognition of longer sequences by individual domains is achieved by expanding the classical KH fold with additional secondary structure elements that elongate the RNA recognition surface [17,18]. The best studied of these expanded KH folds is the Signal Transduction and Activation of RNA (STAR) fold, which mediates RNA recognition in a family of proteins important to splicing, mRNA shuttling and translational repression (Figure 3a) [8,19-21].…”

RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

“…To monitor pnp operon regulation by PNPase, we used a previously described reporter system consisting of a translational fusion between the 5= region of the rpsO-pnp operon, including the first 61 codons of pnp (Fig. 1), and the reporter gene lacZ carried by the transducing GF2 phage (14,31,32). Single ⌬pnp and double ⌬pnp ⌬rnc-38 mutants were lysogenized with GF2 and transformed by pBAD24 plasmid vector derivatives harboring pnp (or pnp mutants as described below) under the control of the arabinose-inducible promoter araBp.…”