Structural and mechanistic studies on Staphylococcal RNA degrading enzymes and multienzyme complexes

Abstract

RNA degrading enzymes and multi-enzyme complexes govern a variety of cellular processes. The role of these enzymes and multi-enzyme assemblies has been suggested to govern gene expression levels in bacteria with modulations leading to a so-called phenotypic switch from the persistent (biofilm forming) to a virulent phase. The role of these enzymes in modulating RNA-dependent signal transduction is less understood although several studies implicate these enzymes in regulating the intracellular levels of RNA messengers. Given the multi-functional roles of these enzymes from housekeeping functions such as RNA recycling to mediating bacterial cell phenotypes, understanding these proteins and multi-enzyme complexes is essential to understand bacterial physiology. The work reported in this thesis represents one step in on-going studies to understand the cellular and mechanistic triggers that enable the assembly of multi-enzyme complexes to degrade RNA. Structural and biochemical characterization of individual components provide an insight into the rationale for a multienzyme assembly. These multi-enzyme complexes, also referred to as the degradosome, have been suggested to be context dependent sequestration of RNA modification and degrading enzymes under specific cellular or environmental cues. These enzymes and complexes have also been demonstrated to influence the phenotype in Staphylococci. Biochemical and structural features of three components of this complex viz., PNPase, RNase J1 and RNase J2 are described in this thesis. This thesis is organized as follows: Chapter 1 provides an introduction to RNA metabolism and degradation in prokaryotes. One section of this chapter provides a compilation of literature describing structural and biochemical features of enzymes associated with the degradosome in bacteria. This description is designed to place the lacunae in our understanding of the RNA degradation process alongside the progress achieved in the characterization of individual component enzymes. A brief description of the degradosome complex is also provided to highlight the differences between Gram-positive and Gram-negative bacteria as well as the components in different bacterial species. This is followed by an introduction to the role of RNA mediated processes in Staphylococci to place the work described in this thesis in the broad context of this bacterial pathogen. The emphasis here is on the bacterial phenotype, in particular the virulent phase characterized by secretion of multiple exotoxins. Finally, a section on reported differences in the degradosome components between Staphylococci and other bacteria is compiled to phrase the broad research questions in this area and the aim and scope of the work described in this thesis. The structure and biochemical features of PNPase are described in Chapter 2. Polynucleotide phosphorylase catalyzes both 3'-5' exoribonuclease and polyadenylation reactions. The crystal structure of Staphylococcus epidermidis PNPase revealed a bound phosphate in the PH2 domain of each protomer coordinated by three adjacent serine residues. Mutational analysis revealed that phosphate coordination by these serine residues was essential to maintain the catalytic center in an active conformation. We note that PNPase forms a complex with RNase J1 and RNase J2 without substantially altering either exoribonuclease and polyadenylation activity of this enzyme. This decoupling of catalytic activity from proteinprotein interactions suggests that association of these endo- or exo-ribonucleases with PNPase could be more relevant for cellular localization or concerted targeting of structured RNA for recycling. There are two RNase J paralogues in Staphylococci- RNase J1 and RNase J2. The structural and biochemical features of these two enzymes and the characterization of the interactions between these two RNase J components in vitro is described in Chapter 3. A comparison of these enzymes with previously described RNase J homologs reveals distinct features that provide a potential rationale for the existence of these RNase J paralogs (in most Gram-positive bacteria). Enzyme assays were performed to determine the relative endo- and exoribonuclease activity of RNase J1 and RNase J2. We note that these activities rely on a metal ion at the active site. An analysis of this metal co-factor and its implication for the reaction mechanism is described in this chapter. Structural studies provided another perspective to the role of these enzymes and rationale for association (the RNase J1- RNase J2 complex). While the overall structures of these enzymes are broadly similar, differences in the active site suggest that the two paralogues might adopt different reaction mechanisms. These studies thus suggest a need to revisit a prevailing hypothesis that RNase J is a functional homologue of E. coli RNase E despite poor sequence similarity. Chapter four provides a summary of biochemical information obtained on the three RNA degrading enzymes. These results suggest a link between the regulation of these enzymes and their assembly in vivo. Indeed, this study, as well as other reports in this area, suggest substantial interactions between multi-enzyme RNA-degrading complex and the biochemical pathways that govern energy metabolism. In the case of PNPase, for example, both citrate and ATP influence PNPase activity. While the association between multiple signal transduction pathways and RNA recycling is not surprising, further work to establish conditional correlation between these intracellular networks is essential to understand these mechanisms. The work reported in this thesis also provides a framework for the identification of target mRNA that might bind the multienzyme RNA degradation complex. The structural and biochemical information of all degradosome components (of which three have been described in this thesis) would substantially aid in recreating the assembly of this multi-enzyme complex in vitro