The role of the S1 domain in exoribonucleolytic activity: Substrate specificity and multimerization

Abstract

RNase II is a 39-59 exoribonuclease that processively hydrolyzes single-stranded RNA generating 59 mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 39-59 exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.

Analysis of the human polynucleotide phosphorylase (PNPase) reveals differences in RNA binding and response to phosphate compared to its bacterial and chloroplast counterparts

“…In order to determine the binding curve, RNA-binding of the purified protein was analyzed in a UV-cross-linking assay using increasing amounts of PNPase. As shown in Figure 6A, while the E. coli enzyme displayed a saturation binding curve with a K d of 11 nM, in agreement with previous reports (BermudezCruz et al 2002;Schubert et al 2004;Stickney et al 2005;Amblar et al 2007), an apparent sigmoid curve, typical of cooperative binding, was observed for the human enzyme. The K d observed for the human PNPase was z16 nM.…”

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

“…Instead, these enzymes have a specific RNA chain size limit that, once approached, causes the release of the remaining RNA chain. As an example, the released chain lengths produced by RNase II and RNase R were found to be 3-5-mers (Cheng and Deutscher 2002) or 4-6-mers (Amblar et al , 2007, and 2-3-mers (Cheng and Deutscher 2002;Vincent and Deutscher 2006) or 1-2-mers (Amblar et al 2007), respectively. The mechanistic basis for this size limit was explained nicely by structural insights provided by the example of RNase II, where binding of a relatively long RNA molecule is important for stabilizing the catalytic complex (Cheng and Deutscher 2002;Frazao et al 2006;Vincent and Deutscher 2006;Amblar et al 2007).…”

PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins

“…PNPase is a phosphate-dependent exonuclease that acts as a trimer (Pruijn 2005). The recent available structure of RNase II highlighted the mechanism of degradation of this hydrolytic enzyme (Amblar et al , 2007Frazão et al 2006;Zuo et al 2006). Homologs of these exonucleases exist in eukaryotic exosomes (a multiprotein complex of exonucleases), and they were shown to be essential in the processing of noncoding RNAs (Houseley et al 2006).…”