Positive regulators of Bacteroides CTnDOT excision and transfer
type: CreativeWork
description: Tetracycline stimulates both excision and transfer of a Bacteroides Conjugative transposon CTnDOT. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the xis2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, I sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains, and the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. I made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of xis2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of PE. The same alanine RteC mutants were used to test the effect of mutations on binding, and all mutants lost binding to PE region. The upstream region of xis2c contained two inverted repeat half-sites. Mutational analysis of these half-sites showed that both half-sites are important for activity. In vivo GUS assays were also done to test whether these half-sites are important for binding. Disrupted inverted repeat sequences resulted in loss of binding to PE region. Thus, I have identified the DNA binding portion of RteC and DNA site to which it binds. The tetracycline regulated excision operon, containing xis2c, xis2d, and exc of the Bacteroides conjugative transposon CTnDOT has been shown to mediate not only the excision of CTnDOT and but also the regulation of transcription of a large operon containing genes required for transfer of the CTnDOT transfer intermediate. In this study, the importance of two excision proteins (Xis2c and Xis2d) was examined to determine their roles in regulating the transc…
abstract: Tetracycline stimulates both excision and transfer of a Bacteroides Conjugative transposon CTnDOT. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the xis2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, I sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains, and the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. I made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of xis2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of PE. The same alanine RteC mutants were used to test the effect of mutations on binding, and all mutants lost binding to PE region. The upstream region of xis2c contained two inverted repeat half-sites. Mutational analysis of these half-sites showed that both half-sites are important for activity. In vivo GUS assays were also done to test whether these half-sites are important for binding. Disrupted inverted repeat sequences resulted in loss of binding to PE region. Thus, I have identified the DNA binding portion of RteC and DNA site to which it binds. The tetracycline regulated excision operon, containing xis2c, xis2d, and exc of the Bacteroides conjugative transposon CTnDOT has been shown to mediate not only the excision of CTnDOT and but also the regulation of transcription of a large operon containing genes required for transfer of the CTnDOT transfer intermediate. In this study, the importance of two excision proteins (Xis2c and Xis2d) was examined to determine their roles in regulating the transc…
author: Park, Ji-Yeon
language: en
keywords: Bacteroides, CTnDOT, Gene regulation
date published: 2012-02-01T00:54:03Z
date modified: 2026-07-22T22:25:27Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/29526