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Genetic analysis of allosteric regulation in bacterial transcriptional activators RhaS and RhaR
Hill, Latavia Keyana
Hill, Latavia Keyana
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Abstract
RhaS and RhaR are positive regulators of the Escherichia coli L-rhamnose regulon and are members of the AraC/XylS family of bacterial proteins. In the presence of L-rhamnose, RhaR activates the expression of rhaSR, thus autoregulates its own expression and activates the expression of RhaS. Once sufficient RhaS protein accumulates, RhaS activates the expression of rhaBAD and rhaT to facilitate L-rhamnose catabolism and transport into the cell, respectively. RhaS and RhaR proteins each contain two domains: C-terminal DNA-binding domains (DBD) and N-terminal regulatory domains (NTD). The DBDs are responsible for DNA-binding and transcription, while the NTDs are responsible for binding their common effector, L-rhamnose, and dimerization. These two domains are connected by a flexible linker that has thus far been shown to play no additional roles in the functions of RhaS or RhaR [1]. The major foci of the research presented in this dissertation are as follows: determine and analyze high-resolution crystal structures of the RhaR NTD, elucidate the role of DBD residues T270-D282 in RhaR allostery, identify L-rhamnose binding residues in RhaS, and to characterize the role of N-terminal arm residues in RhaS allostery. Crystal structure analysis of the RhaR NTD revealed the presence of a metal in the L-rhamnose binding pocket and also determined that the V22-T35 region was disordered only in the apo structure. Alanine mutagenesis experiments determined that RhaR variants R25A and F31A exhibited increased activity in the absence of L-rhamnose, which led to the hypothesis that residues in the V22-T35 region make an interdomain contact with the DBD to inhibit the protein’s activity in the absence of L-rhamnose. My results determined that RhaR DBD variants T270A-D282A exhibited decreased activity in the absence of L-rhamnose, in contrast to variants R25A and F31A. This finding suggests that residues T270-D282 likely do not contact the V22-T35 NTD region in the absence of L-rhamnose to hold RhaR in its non-activating state. RhaS activate transcription to maximal levels in the presence of L-rhamnose. In AraC, residues involved in L-arabinose were previously identified [2]. The results presented here suggests that RhaS residues V17, Q25, H31, H71, and Y73 participate in L-rhamnose binding and/or allosteric signaling and also suggest that similar to RhaR, RhaS likely binds a metal. We previously determined that RhaS N-terminal arm deletion variants exhibited decreased activity in the presence of L-rhamnose, which suggested that the mechanism of RhaR allostery differs than that of AraC [3]. The results presented here determined that RhaS residues L4, D8, F9, and F10 are likely involved in increasing the protein’s activity in the presence of L-rhamnose.
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Date
2022-12-31
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University of Kansas
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Keywords
Microbiology, Allostery, Escherichia coli, L-rhamnose, RhaR, RhaS, Transcription