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PMID: 10940041 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Genetic evidence that transcription activation by RhaS involves specific amino acid contacts with sigma 70.

Journal of bacteriology ·Vol. 182 ·No. 17 ·2000-09-00 ·Pages 4959-69

Bhende PM, Egan SM

Abstract

RhaS activates transcription of the Escherichia coli rhaBAD and rhaT operons in response to L-rhamnose and is a member of the AraC/XylS family of transcription activators. We wished to determine whether sigma(70) might be an activation target for RhaS. We found that sigma(70) K593 and R599 appear to be important for RhaS activation at both rhaBAD and rhaT, but only at truncated promoters lacking the binding site for the second activator, CRP. To determine whether these positively charged sigma(70) residues might contact RhaS, we constructed alanine substitutions at negatively charged residues in the C-terminal domain of RhaS. Substitutions at four RhaS residues, E181A, D182A, D186A, and D241A, were defective at both truncated promoters. Finally, we assayed combinations of the RhaS and sigma(70) substitutions and found that RhaS D241 and sigma(70) R599 met the criteria for interacting residues at both promoters. Molecular modeling suggests that sigma(70) R599 is located in very close proximity to RhaS D241; hence, this work provides the first evidence for a specific residue within an AraC/XylS family protein that may contact sigma(70). More than 50% of AraC/XylS family members have Asp or Glu at the position of RhaS D241, suggesting that this interaction with sigma(70) may be conserved.

MeSH Terms
Aldose-Ketose Isomerases/genetics Amino Acid Substitution Amino Acids Base Sequence DNA-Binding Proteins/chemistry,genetics,metabolism DNA-Directed RNA Polymerases/genetics,metabolism Escherichia coli/genetics Escherichia coli Proteins Gene Expression Regulation, Bacterial Membrane Transport Proteins/genetics Models, Molecular Molecular Sequence Data Phosphotransferases (Alcohol Group Acceptor)/genetics Protein Conformation Sigma Factor/genetics,metabolism Symporters Trans-Activators/chemistry,genetics,metabolism Transcriptional Activation
Chemicals
Amino Acids DNA-Binding Proteins Escherichia coli Proteins Membrane Transport Proteins RhaS protein, E coli RhaT protein, E coli Sigma Factor Symporters Trans-Activators Phosphotransferases (Alcohol Group Acceptor) rhamnulokinase RNA polymerase sigma 70 DNA-Directed RNA Polymerases Aldose-Ketose Isomerases L-rhamnose isomerase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Bhende P M
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Egan S M
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.182.17.4959-4969.2000
Published
2000-09-00
Pages
4959-69
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC111377
Subset
IM
Grants
NIGMS NIH HHS · R01 GM055099 · United States
NIGMS NIH HHS · R29 GM055099 · United States
NIGMS NIH HHS · GM55099 · United States
External Links
PubMed source DOI: 10.1128/JB.182.17.4959-4969.2000 PMC Full Text View journal details
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