AbstractThe aim of this thesis was to expand the understanding of sRNA-mediated post-transcriptional gene regulation by biophysically and biochemically analyzing the interplay of small non-coding RNAs, the RNA chaperone, Hfq, and the endoribonuclease, RNase E. To address this, three important questions were considered: Is the C-terminal region of Hfq important? Is Hfq the only regulator of sRNA function? Do the roles of Hfq differ towards sRNAs involved in up-regulation versus those involved in down-regulation of their target mRNAs?
Firstly, the importance of the C-terminal region in Hfq was investigated. In
some bacteria, Hfq has evolved to contain an extended C-terminal region, but the reason for this has always been unclear. Therefore, characterization of Hfq from the model organism, E. coli, which contains a very long C-terminal region, was compared with the pathogen, V. cholerae, which contains a shorter C-terminal region. The work presented identifies that the longer C-terminal region of E. coli causes the protein to be more stable than Hfq from the V. cholerae Hfq. It is inferred that the increase in stability observed is generated from the C-terminal region interacting with, and protecting the subunit interface from denaturation. However, it remains unknown as to why such bacterial species have evolved extended CTRs with differing stabilizing effects.
Secondly, to assess if Hfq is the only regulator of sRNA function, its role in the MicA sRNA-mediated regulation of its mRNA target, ompA, was investigated. The work presented identifies Hfq to be critical for efficient regulation, causing structural changes to the MicA that expose the region of complementarity to ompA, and thus resulting in enhanced MicA:ompA pairing. However, Hfq was demonstrated not to be the only regulator of MicA function as it was also found that MicA forms self-inhibitory dimers at specific concentrations in the presence of Mg2+. The finding that sRNAs can sense their surrounding environments, with particular reference to ion levels, identifies a hitherto unknown mechanism of gene regulation.
Thirdly, to assess if the role of Hfq differs towards sRNAs that up-regulate their mRNA targets, versus sRNAs that down-regulate their mRNAs targets, the interactions between RprA (an sRNA involved in up-regulation) and OxyS (an sRNA involved in down-regulation) with Hfq were compared. The work presented indicates the functional roles of Hfq to be similar with EMSA analysis showing that Hfq provides a platform for both sRNAs and mRNA to bind simultaneously. In addition, by determining the first solution structures of these complexes, it could be seen that Hfq changes the structures of both OxyS and RprA. However, whilst these affects result in both sRNAs having an increased level of binding to their mRNA targets, it has been found that these interactions with Hfq cause their relative susceptibilities to RNase E degradation to alter. For OxyS (involved in down-regulation), an enhanced rate of degradation by RNase E has been observed, whilst for RprA (involved in up-regulation), Hfq protected the sRNA from RNase E degradation. These findings indicate that although the fundamental roles of Hfq are similar, the functional outcomes can be different and appear to be dependent on the type of regulation the sRNA encodes.
|Date of Award||Mar 2012|
|Supervisor||Anastasia Callaghan (Supervisor) & Geoff Kneale (Supervisor)|