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Specific residues and regions within the KH and S1 domains of PNPase that are important for RNA binding and autoregulation

University of British Columbia

The turnover of RNA is a critical process governing key aspects of cell growth, regulation, and survival. In particular, gene expression is largely mediated by the differential stabilities of mRNAs and their corresponding levels of translation. Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3’ exoribonuclease found in many different organisms. Although PNPase functions are diversified in nature, in E. coli, it functions primarily in mRNA degradation by 3’ to 5’ phosphorolysis. It is also a major component of the RNA degradosome – a macromolecular association of proteins involved in RNA decay. The six N-terminal RNase PH-like domains of PNPase assemble into a ring-shaped trimer forming the active core, through which the RNA substrate is threaded during degradation. The C-terminal KH and S1 RNA-binding domains are positioned at the entrance to this core structure and have been shown to facilitate substrate recognition and interaction. Towards developing a better understanding of the proposed KH-S1 binding platform, we have endeavoured to identify and investigate key residues in these domains responsible for RNA binding. In this study, we use a convenient pnp::lacZ fusion reporter strain to assess autoregulatory ability by both PNPase lacking the KH and S1 domains and containing point mutations in these domains. PNPase represses its own mRNA resulting in autoregulation and RNA binding has been shown to facilitate this. KH and S1 point mutants identified from a library and those predicted to contact RNA were expressed in the reporter strain. Mutants found to be deficient in autoregulation poorly repressed levels of pnp::lacZ transcript compared to WT PNPase, resulting in higher β-galactosidase levels. Interesting mutants were purified and studied using in vitro band-shift and phosphorolysis assays to assess binding and enzymatic activity. We show that reductions in substrate affinity accompanied impairment in PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting ability to autoregulate, and apparent Kd values reflecting in vitro affinities for a model RNA substrate, implicating the KH and S1 domains of PNPase, and specific residues within them, in substrate binding and autoregulation. Our findings are discussed in the context of PNPase structure, binding architecture, and function.

Text

2012-08-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/43127

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/