Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 384, Issue 5, Pages 1037-1047Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.10.011
Keywords
TodT; Pseudomonas; response regulator; two-component systems; sensor kinases
Categories
Funding
- Spanish Ministry of Science and Education [CICYT BIO-2006-05668, BFU 2005-0487-C02-02, CSD 2007-00010]
- Junta de Andalucia [CIV344, CIV1912]
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The TodS/TodT two-component system controls the expression of nod genes for toluene degradation in Pseudomonas putida. TodT binds to two pseudopalindromes at -106 (Box-1) and -85 (Box-2), as well as to a half-palindrome (Box-3), with respect to the main transcription initiation site in the P-todX promoter. TodT recognizes each half-palindrome in Boxes-1 and -2, but affinities for these sequences are lower than those for the pseudopalindromes, pointing towards positive cooperativeness in intrabox recognition. TodT's affinity for DNA fragments containing two vicinal boxes (either Boxes-1. and -2 or Boxes-2 and -3) is higher than its affinity for individual boxes, suggesting interbox cooperativeness. Similar patterns of cooperativeness were observed for the recombinant TodT DNA-binding domain [C-terminal TodT fragment (aa 154-206) (C-TodT)], suggesting important cooperativeness determinants in this domain. Occupation of P-todX by TodT is initiated at Box-1, and optimization of its palindromic order increases affinity in vitro; however, this does not result in enhanced in vivo gene expression. Mutations at either half of the Box-1 palindrome have no significant effects on transcriptional activity, whereas mutations in the entire Box-1 cause a 12-fold reduction. Using atomic force microscopy, we show that TodT induces a DNA hairpin bend at P-todX between Boxes-2 and -3, as supported by footprint studies showing a hyperreactive nucleotide at G -68. The N-terminal part of TodT seems to play a central role in hairpin formation, since C-TodT neither induces a bend nor causes G -68 hyperreactivity in footprints. This hairpin seems important for transcriptional activation, since C-TodT binding to P-todX does not stimulate transcription. (C) 2008 Elsevier Ltd. All rights reserved.
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