Thermodynamic Parameters of Specific and Nonspecific Protein-DNA Binding

Baichoo

Journal of Biological Chemistry

2001

The ␣-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the ␣-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The ␣ dimer and DNA formed a 1:1 complex in solution. Complex formation at 25°C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (⌬c p ) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the ␣-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between ␣-subunit and DNA was not formed.A typical Escherichia coli promoter is identified by two conserved DNA sequences, the Ϫ10 and Ϫ35 regions, separated by a spacer with a consensus length of 17 bp (reviewed in Ref. 1). In addition to these core promoter elements many promoters contain an A-T-rich region located upstream of the Ϫ35 region (reviewed recently in Ref.2). This so-called UP element was discovered first in promoters for ribosomal RNA where it was recognized that the UP element increased transcription from these promoters in a factor-independent fashion, suggesting its direct effect on RNA polymerase (3). The ␣-subunit of RNA polymerase was subsequently identified as a target for a direct contact with the UP element (4). Mutations of the ␣-subunit defective in UP element binding were found in the C-terminal domain of the ␣-subunit, identifying two regions (vicinity of residues 265 and 298, respectively) in this domain as important for UP element recognition (5 and 6). The C-terminal DNAbinding domain of ␣ is attached to the N-terminal domain of the protein by a long, flexible linker (7 and 8).1 Site selection experiments were used to identify the consensus UP element sequence (9). Further studies revealed that the UP element consists of two subsites, the proximal and the distal site, and each of these sites binds one monomer of the ␣-subunit (10). Fine mapping of the ␣-subunit-UP element interactions revealed that the ␣-subunit interacted with the bases in the minor groove and with the DNA backbone along the minor groove. The interactions with the major groove were found to be minimal (11).The mechanism ...

Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Interaction of the α-Subunit of Escherichia coliRNA Polymerase with DNA

Baichoo

Journal of Biological Chemistry

2001

Bioorganic &Amp; Medicinal Chemistry

“…Yet freeenergy prediction is complicated by the ability of enthalpy and entropy, which usually move in opposite directions, to compensate for changes in order to maintain fairly stable overall free energies of binding. 23,33 Compensation occurs frequently in biological systems, for enthalpy and entropy values can vary widely in dependent fashion, even though ÁG values cluster in a relatively narrow range. 23 A potential source of unfavorable enthalpy change is from strain, often arising from energetically costly DNA distortions; crystal structures show that GCN4 binds to AP-1 DNA with no distortion, 5 but the GCN4-ATF/CREB structure shows that the central base pairs exhibit A-form character and a 20 bend.…”

Section: Resultsmentioning

confidence: 99%

The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif

Kise

Shin

2001

Abstract-We have used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) to short DNA duplexes, in which thymines were replaced with uracils, in order to quantify the contributions of the C5 methyl group on thymines with alanine methyl side chains. We simplified the a-helical GCN4 bZIP by alanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. Titration of fluorescein-labeled duplexes with increasing amounts of protein yielded dissociation constants in the low-to-mid nanomolar range for all bZIP mutants in complex with the AP-1 target site (5 0 -TGACTCA-3 0 ); binding to the nonspecific control duplex was > 1000-fold weaker. Small changes of <1 kcal/mol in binding free energies were observed for wild-type bZIP and 4A mutant to uracil-containing AP-1, whereas 11A and 18A bound almost equally well to native AP-1 and uracil-containing AP-1. These modest changes in binding affinities may reflect the multivalent nature of protein-DNA interactions, as our highly mutated proteins still exhibit native-like behavior. These protein mutations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to that of the native protein-DNA complex. #

Wiley Encyclopedia of Chemical Biology

Receptor–Ligand Interactions in Biological Systems

Meroueh

2008

Despite the significant research that has been invested in understanding molecular recognition in biological systems, accurate prediction of macroscopic properties based on microscopic interactions remains elusive, which makes it difficult to identify systematically tight binding inhibitors in computational drug design. In the past, most ligand design efforts have centered on descriptors derived mainly from structure, neglecting entropic effects that develop from receptor flexibility. The lack of explicit incorporation of receptor motion has meant that compensatory effects between enthalpy and entropy, which are essential for the accurate estimation of free energy, have also been neglected. In addition, cooperative effects, which develop from alteration in the motion of the receptor because of ligand binding, are not captured. These effects pose a challenge for the design of small molecules that allosterically modulate protein‐protein and protein‐small‐molecule interactions. Here, we describe macroscopic properties of receptor‐ligand interactions, which are followed by a discussion of how these properties are predicted with microscopic interactions. A discussion then ensues on some fundamental aspects of the molecular recognition process that have attracted renewed attention, such as conformational selection versus induced‐fit binding, entropy‐enthalpy compensation, and allostery.