Flexibility and Adaptability in Binding of E. coli Cytidine Repressor to Different Operators Suggests a Role in Differential Gene Regulation

Tretyachenko-Ladokhina, Vira; Cocco, Melanie J.; Senear, Donald F.

doi:10.1016/j.jmb.2006.06.085

Cited by 20 publications

(32 citation statements)

References 45 publications

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“…CytR binding to its small molecule effector (cytidine) has no effect on intrinsic DNA binding affinity ( e.g. [56,57••]). Instead, the cytidine-induced conformational change disallows simultaneous CytR contacts with CRP and cytO .…”

Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

“…Instead, the cytidine-induced conformational change disallows simultaneous CytR contacts with CRP and cytO . As a result (i) cooperative DNA binding of CytR and CRP is diminished, allowing RNA polymerase to compete for cytO , and (ii) direct interactions between CRP and RNA polymerase are altered [56, 57••, 58]. …”

Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

“…No high resolution structure has been obtained, but biophysical data suggest that CytR can adopt multiple conformations in the apo-state that are constrained differently when bound to operators with distinct half-site spacings [57••]. Unlike many members of the LacI/GalR family, altered CytR•CRP interactions provide a “rheostatic” rather than “on/off” switching mechanism.…”

Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

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Allostery in the LacI/GalR family: variations on a theme

Swint-Kruse

Matthews

2009

Current Opinion in Microbiology

148

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SummaryThe lactose repressor protein (LacI) was among the very first genetic regulatory proteins discovered, and more than 1000 members of the bacterial LacI/GalR family are now identified. LacI has been the prototype for understanding how transcription is controlled using small metabolites to modulate protein association with specific DNA sites. This understanding has been greatly expanded by the study of other LacI/GalR homologues. A general picture emerges in which the conserved fold provides a scaffold for multiple types of interactions -including oligomerization, small molecule binding, and protein•protein binding -that in turn influence target DNA binding and thereby regulate mRNA production. Although many different functions have evolved from this basic scaffold, each homologue retains functional flexibility: For the same protein, different small molecules can have disparate impact on DNA binding and hence transcriptional outcome. In turn, binding to alternative DNA sequences may impact the degree of allosteric response. Thus, this family exhibits a symphony of variations by which transcriptional control is achieved. Overview of the LacI/GalR familyIn virtually all bacteria, LacI/GalR family members regulate transcription for a wide range of processes. First catalogued in 1992 by Weickert and Adhya [1], sequences of >1000 characterized and hypothetical homologues are now known (2008 BLAST search of SwissProt). These proteins have not been found in archaebacteria or eukaryotes, although proteins with homologous domains are ubiquitous.The LacI/GalR family can be divided into >33 paralogue groups that appear to derive from an ancestral gene. As many as 22 paralogues co-exist in a single species. Many members coordinate available nutrients with expression of catabolic genes [1], but some regulate processes as diverse as nucleotide biosynthesis and toxin expression (e.g. [2,3]). Two members are "master" regulators: homologues CcpA and CRA control expression of enzymes that determine carbon flow in Gram-negative and Gram-positive bacteria, respectively. If these key proteins are disabled, virulence is altered in several pathogens (e.g. [4,5•,6•,7]).The common function of the LacI/GalR proteins, which features allosteric regulation of DNA binding to modulate transcription, is shown in Figure 1. Each homologue has evolved a unique variation: In addition to binding specific "operator" DNA sequences, each protein exhibits specificity for distinct effector ligands. Although most members repress transcription, someCorresponding Author: Swint-Kruse, Liskin (E-mail: lswint-kruse@kumc.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, a...

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Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

Section: Functional and Allosteric Variation Within The Laci/galr Familymentioning

confidence: 99%

See 1 more Smart Citation

Allostery in the LacI/GalR family: variations on a theme

Swint-Kruse

Matthews

2009

Current Opinion in Microbiology

148

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“…The unfolded linkers in CytR allow its two N-terminal DNA-binding domains to bind operators with varied half-site spacing (Fig. 1E) (28). Notably, the disordered linkers do not propagate allosteric information to the DNA-binding domains as found for LacI.…”

Section: Laci/galr Proteinsmentioning

confidence: 95%

“…For high affinity DNA binding, CytR requires cooperative binding of flanking catabolite repressor proteins (CRPs) (10,28). The unfolded linkers in CytR allow its two N-terminal DNA-binding domains to bind operators with varied half-site spacing (Fig.…”

Section: Laci/galr Proteinsmentioning

confidence: 99%

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

Bondos

Swint-Kruse

Matthews

2015

Journal of Biological Chemistry

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Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: Bioinformatics‐based predictions generate true positives and false negatives

2008

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In protein families, conserved residues often contribute to a common general function, such as DNA-binding. However, unique attributes for each homologue (e.g. recognition of alternative DNA sequences) must arise from variation in other functionally-important positions. The locations of these “specificity determinant” positions are obscured amongst the background of varied residues that do not make significant contributions to either structure or function. To isolate specificity determinants, a number of bioinformatics algorithms have been developed. When applied to the LacI/GalR family of transcription regulators, several specificity determinants are predicted in the 18 amino acids that link the DNA-binding and regulatory domains. However, results from alternative algorithms are only in partial agreement with each other. Here, we experimentally evaluate these predictions using an engineered repressor comprising the LacI DNA-binding domain, the LacI linker, and the GalR regulatory domain (LLhG). “Wild-type” LLhG has altered DNA specificity and weaker lacO1 repression compared to LacI or a similar LacI:PurR chimera. Next, predictions of linker specificity determinants were tested, using amino acid substitution and in vivo repression assays to assess functional change. In LLhG, all predicted sites are specificity determinants, as well as three sites not predicted by any algorithm. Strategies are suggested for diminishing the number of false negative predictions. Finally, individual substitutions at LLhG specificity determinants exhibited a broad range of functional changes that are not predicted by bioinformatics algorithms. Results suggest that some variants have altered affinity for DNA, some have altered allosteric response, and some appear to have changed specificity for alternative DNA ligands.

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Flexibility and Adaptability in Binding of E. coli Cytidine Repressor to Different Operators Suggests a Role in Differential Gene Regulation

Cited by 20 publications

References 45 publications

Allostery in the LacI/GalR family: variations on a theme

Allostery in the LacI/GalR family: variations on a theme

Flexibility and Disorder in Gene Regulation: LacI/GalR and Hox Proteins

Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: Bioinformatics‐based predictions generate true positives and false negatives

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