Document Detail


Probing the physical basis for trp repressor-operator recognition.
MedLine Citation:
PMID:  10092458     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The bacterial repressor protein, trp repressor, is one of the best studied transcriptional regulatory proteins in terms of function, structure, dynamics and stability. Despite these significant advances, the structural and energetic basis for the specific recognition of its operator sites by trp repressor remains poorly understood. In fact, recognition in this system is controled by the binding of the co-repressor ligand, l-tryptophan, as well as by conformational and dynamic properties of the operator targets, DNA sequence-dependent control of the oligomerization properties of the repressor, water-mediated interactions, and specific interactions involving the peptide backbone and phosphate moieties. Moreover, only one direct contact between the protein and the DNA is evident from the crystallographically determined structure of the complex. In an attempt to better define how the various sequence elements in the operator target contribute to this complex control of affinity and cooperativity of trp repressor binding, we have studied the binding of trp repressor to a series of mutated operator targets using fluorescence anisotropy, which provides very high quality data allowing fairly precise estimations of the affinities involved. We conclude from these studies that even on very small (25 bp) targets, the repressor binds slightly cooperatively, populating a 2:1 dimer/DNA complex, and then at higher concentrations a third dimer is bound with significantly lower affinity, revealing an inherent asymmetry in the trpEDCBA-derived target. Investigation of the basis for the asymmetry implicates the identity of the second base in the so-called structural half-site GNACT, which apparently influences the switch between tandem and simple binding. Mutation of the C or the T bases in the structural half-site abolishes all specificity in binding, and alteration of the single direct contact, the G of the structural half-site, or the central TTAA significantly lowers the affinity of the dimer for its site, without modifying the apparent cooperativity. Finally, we note that the order of affinity is conserved in the absence of the co-repressor, and moreover, it is in all cases significantly higher than that observed for holo-repressor binding to non-specific DNA, indicating that one cannot simply equate apo-repressor and non-specific binding.
Authors:
A O Grillo; M P Brown; C A Royer
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of molecular biology     Volume:  287     ISSN:  0022-2836     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  1999 Apr 
Date Detail:
Created Date:  1999-06-07     Completed Date:  1999-06-07     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  539-54     Citation Subset:  IM    
Copyright Information:
Copyright 1999 Academic Press.
Affiliation:
School of Pharmacy, University of Wisconsin-Madison, 425 N. Charter, Madison, WI, 53706, USA.
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MeSH Terms
Descriptor/Qualifier:
Bacterial Proteins / chemistry*,  genetics,  metabolism*
Base Sequence
Binding Sites / genetics
DNA, Bacterial / chemistry,  genetics,  metabolism
Escherichia coli / genetics,  metabolism
Helix-Turn-Helix Motifs
Macromolecular Substances
Models, Biological
Models, Molecular
Mutation
Nucleic Acid Conformation
Operator Regions, Genetic*
Protein Binding
Protein Conformation
Repressor Proteins / chemistry*,  genetics,  metabolism*
Thermodynamics
Tryptophan / metabolism
Grant Support
ID/Acronym/Agency:
GM39969/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Bacterial Proteins; 0/DNA, Bacterial; 0/Macromolecular Substances; 0/Repressor Proteins; 0/tryptophan repressor protein; 73-22-3/Tryptophan

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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