| Autor: |
Karney MM; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. monika.karney@unlv.edu., McKenna JA; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. joy.immak@unlv.edu., Weatherspoon-Griffin N; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. nweatherspoon@gmail.com., Karabachev AD; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. Alexander.Karabachev@med.uvm.edu., Millar ME; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. makensiem@gmail.com., Potocek EA; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. potocek.e@gmail.com., Wing HJ; School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV 89154-4004, USA. helen.wing@unlv.edu. |
| Abstrakt: |
The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB . Our previous in vivo studies at a different VirB-dependent promoter, icsP , found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB , virF , spa15 , and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed. |
