Talk on Friday 03:45-04:00pm submitted by Margo Gebbie
A Riboswitch-Containing sRNA Controls Gene Expression by Sequestration of a Response Reg
Margo P Gebbie (Cell Biology and Molecular Genetics, University of Maryland), Sruti Debroy (Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston), Jonathan Goodson (Cell Biology and Molecular Genetics, University of Maryland), Arati Ramesh (Biochemistry, University of Texas Southwestern Medical Center at Dallas), Danielle Garsin (Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston), Wade Winkler (Cell Biology and Molecular Genetics, University of Maryland)
Adenosyl cobalamine (AdoCbl) is required for the metabolism of ethanolamine (EA) in Enterococcus faecalis, both as an enzyme co-factor and for the induction of the EA utilization (eut) genes. It acts through an AdoCbl-binding riboswitch to induce the eut genes, but the mechanism of control is incompletely understood. Gene expression also requires EA, and this compound activates a two-component system composed of the sensor kinase, EutW, and its cognate response regulator, EutV, by phosphorylation. Active EutV is an antiterminator that binds nascent transcripts by recognizing a dual hairpin substrate and preventing terminator formation. Previously, we found that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual hairpin substrate for EutV. From this, we proposed a model where, in the absence of AdoCbl, EutX sequesters EutV in an inactive complex. In contrast, when AdoCbl is in excess, its binding to the riboswitch prevents EutX/EutV complex formation, freeing EutV to induce gene expression by antitermination. This model assumes that the EutX-EutV interaction is preferred relative to the four different EutV antitermination sites within the eut gene cluster. Therefore, to explore how EutX is able to successfully sequester EutV in vivo, we have purified variants of EutV and EutX and investigated their interactions through various binding assays in vitro. These data suggest that EutX may contain multiple EutV binding sites, a feature that we hypothesize is likely to improve affinity of EutV for EutX, such that it can outcompete the EutV antitermination sites. These data provide important insight into the molecular mechanism of a protein-sequestering sRNA.
DebRoy, S., Gebbie, M., Ramesh, A., Goodson, J., Cruz, M., van Hoof, A., Winkler, W., and Garsin, D. Science. A Riboswitch-containing sRNA Controls Gene Expression by Sequestration of a Response Regulator. August 2014: 345 (6199), 937-940.
Keywords: Riboswitch, Two Component System, RNA Regulation