Calorimetric analysis of lambda cI repressor binding to DNA operator sites.


Abstract

Enthalpies and heat capacities were determined by isothermal titration calorimetry for bacteriophage lambda cI repressor binding to DNA containing various combinations of the three operator sites OR1, OR2, and OR3 (each comprising a consensus half-site and a specific nonconsensus half-site). Differential scanning calorimetry was employed to evaluate the effects of specific DNA binding on thermal melting of the N-terminal and C-terminal repressor domains. Principal findings of this study are as follows: (1) Binding of repressor to each of the DNA operators is dominated by a large negative enthalpy, in agreement with earlier van't Hoff analyses of quantitative footprint titration data [Koblan & Ackers (1992) Biochemistry 31, 57-65]. The calorimetric data also revealed negative heat capacities for cI binding that are of comparable magnitude with many other systems [Spolar & Record (1994) Science 263, 777-784]. However, this feature in combination with the large negative values of binding enthalpies leads to an enthalpic dominance throughout the physiological temperature range. The resulting thermodynamic profile is opposite to the entropically dominated binding observed for many systems, including lambda cro repressor which binds to the same sites as cI and also employs a helix-turn-helix binding domain [Takeda et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8180-8184]. It is suggested that these thermodynamic differences may arise from interactions of the cI repressor's N-terminal "arm" with the DNA. (2) Repressor monomers do not bind significantly to DNA containing either a consensus half-site or a nonconsensus half-site. Binding affinity to the double-consensus operator is much weaker than to any of the natural full-site operators. The same was found with other combinations of half-sites. A mutant repressor (PT158) which is severely defective in dimerization [Burz et al. (1994) Biochemistry 33, 8399-8405] was also found to bind only full-site operators and showed dimeric stoichiometry. (3) The thermal melting unit for N-terminal domains in the absence of DNA was found to reach values of 6-8 (monomer units) at concentrations where high-order oligomers of wild-type protein are formed [Senear et al. (1993) Biochemistry 32, 6179-6189]. However, in the presence of DNA operator sites, the cooperative unit for thermal unfolding was reduced to precisely two monomers, indicating that the N-terminal domain binds strictly as a dimer. (4) Significant nonadditivity was observed for the repressor binding enthalpies and heat capacities determined with multiple combinations of full-site operators.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 5076, 5077 Extra Details: operator sites; DNA binding; helix-turn-helix binding domain;,cooperative unit; structural origins

Submission Details

ID: pQ4YTumB3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Merabet E;Ackers GK,Biochemistry (1995) Calorimetric analysis of lambda cI repressor binding to DNA operator sites. PMID:7612597
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3KZ3 2010-02-23 1.64 A structure of a lambda repressor fragment mutant
5ZCA 2018-08-15 1.8 Crystal structure of lambda repressor (1-20) fused with maltose-binding protein
1LMB 1991-11-05 1.8 REFINED 1.8 ANGSTROM CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR-OPERATOR COMPLEX
1F39 2000-07-26 1.9 CRYSTAL STRUCTURE OF THE LAMBDA REPRESSOR C-TERMINAL DOMAIN
3WOA 2015-04-29 2.0 Crystal structure of lambda repressor (1-45) fused with maltose-binding protein
1LLI 1994-08-31 2.1 THE CRYSTAL STRUCTURE OF A MUTANT PROTEIN WITH ALTERED BUT IMPROVED HYDROPHOBIC CORE PACKING
1RIO 2004-01-27 2.3 Structure of bacteriophage lambda cI-NTD in complex with sigma-region4 of Thermus aquaticus bound to DNA
1KCA 2001-12-21 2.91 Crystal Structure of the lambda Repressor C-terminal Domain Octamer
1LRP 1989-01-09 3.2 COMPARISON OF THE STRUCTURES OF CRO AND LAMBDA REPRESSOR PROTEINS FROM BACTERIOPHAGE LAMBDA
3BDN 2008-04-15 3.91 Crystal Structure of the Lambda Repressor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Repressor protein cI P03034 RPC1_LAMBD