Lac repressors

Lac repressor binds to both the major and the minor grooves inducing a sharp bend in the DNA... 

More than 30 years ago Jacob and Monod introduced the Escherichia coli lac operon as a model for gene regulation. The lac repressor molecule functions as a switch, regulated by inducer molecules, which controls the synthesis of enzymes necessary for E. coli to use lactose as an energy source. In the absence of lactose the repressor binds tightly to the operator DNA preventing the synthesis of these enzymes. Conversely when lactose is present, the repressor dissociates from the operator, allowing transcription of the operon. 

The lac repressor monomer, a chain of 360 amino acids, associates into a functionally active homotetramer. It is the classic member of a large family of bacterial repressors with homologous amino acid sequences. PurR, which functions as the master regulator of purine biosynthesis, is another member of this family. In contrast to the lac repressor, the functional state of PurR is a dimer. The crystal structures of these two members of the Lac I family, in their complexes with DNA fragments, are known. The structure of the tetrameric lac repressor-DNA complex was determined by the group of Mitchell Lewis, University of Pennsylvania, Philadelphia, and the dimeric PurR-DNA complex by the group of Richard Brennan, Oregon Health Sciences University, Portland. 

Figure 8.21 Richardson-type diagram of the structure of one suhunit of the lac repressor. The polypeptide chain is arranged in four domains, an amino terminal DNA-hinding domain (red) with a helix-tum-helix motif, a hinge helix (purple), a large core domain which has two subdomains (green and hlue) and a C-terminal a helix. (Adapted from M. Lewis et al.. Science 271 1247-1254, 1996.)...

The polypeptide chain of the lac repressor subunit is arranged in four domains (Figure 8.21) an N-terminal DNA-hinding domain with a helix-turn-helix motif, a hinge helix which binds to the minor groove of DNA, a large core domain which binds the corepressor and has a structure very similar to the periplasmic arablnose-binding protein described in Chapter 4, and finally a C-terminal a helix which is involved in tetramerization. This a helix is absent in the PurR subunit structure otherwise their structures are very similar. 

The tetrameric structure of the lac repressor has a quite unusual V-shape (Figure 8.22). Each arm of the V-shaped molecule is a tight dimer, which is very similar in structure to the PurR dimer and which has the two N-termi-nal DNA binding domains close together at the tip of the arm. The two dimers of the lac repressor are held together at the other end by the four carboxy-terminal a helices, which form a four-helix bundle. 

Figure 8.23 The helix-turn-helix motifs of the subunits of both the PurR and the lac repressor subunits bind to the major groove of DNA with the N-terminus of the second helix, the recognition helix, pointing into the groove. The two hinge helices of each arm of the V-shaped tetramer bind adjacent to each other in the minor groove of DNA, which is wide and shallow due to distortion of the B-DNA structure. (Adapted from M.A. Schumacher et al.. Science 266 763-770, 1994.)...

Boelens, R., et al. Complex of lac repressor headpiece with a 14 base-pair lac operator fragment studied by two-dimensional nuclear magnetic resonance. /. Mol. Biol. 193 213-216, 1987. 

These signals in the NOE spectra therefore in principle make it possible to determine which fingerprint in the COSY spectrum comes from a residue adjacent to the one previously identified. For example, in the case of the lac-repressor fragment the specific Ser residue that was identified from the COSY spectrum was shown in the NOE spectrum to interact with a His residue, which in turn interacted with a Val residue. Comparison with the known amino acid sequence revealed that the tripeptide Ser-His-Val occurred only once, for residues 28-30. 

Regulatory proteins Insulin Somatotropin Thyrotropin lac repressor NEl (nuclear factor 1) Catabolite activator protein (CAP) API... 

A review is given of the application of Molecular Dynamics (MD) computer simulation to complex molecular systems. Three topics are treated in particular the computation of free energy from simulations, applied to the prediction of the binding constant of an inhibitor to the enzyme dihydrofolate reductase the use of MD simulations in structural refinements based on two-dimensional high-resolution nuclear magnetic resonance data, applied to the lac repressor headpiece the simulation of a hydrated lipid bilayer in atomic detail. The latter shows a rather diffuse structure of the hydrophilic head group layer with considerable local compensation of charge density. 

Figure 1. Manual reorientation of a backbone loop in lac-repressor was necessary to satisfy NOE constraints. Thin line original structure thick line modified structure.

Figure 2. Final structure of lac-repressor in solution derived from 2D NMR combined with MD.

Helix-turn-helix j Ecoli i Phage I Mammals lac repressor CAP Xcl, cro, and tryptophan and 434 repressors homeo box proteins Pit-1, Oct1,Oct2... 

F. Culard, M. Schnarr, and J. C. Maurizot, Interaction between the lac operator and the lac repressor headpiece Fluorescence and circular dichroism studies, EMBO J. 1, 1405-1409 (1982). 

In protein molecules with two or more tryptophan residues, it is necessary to obtain first the fluorescence decay curves for the individual residues. For this purpose, additional spectroscopic information is necessary. One can use the dependence of the decay curves on emission wavelength, apply selective fluorescence quenchers, or selectively modify one of the tryptophan residues. The results of Brochon et al. for the lac repressor(44) and those of Beechem et al. for alcohol dehydrogenase(45) provide evidence in favor of such approaches. 

Ha et al. [18] have measured the equilibrium quotients for the formation of a complex between the lac repressor protein and a symmetric operator sequence of DNA as a function of temperature. Their results are given below. (The standard state is 1 mol dm. )... 

In addition, the i gene, which encodes the lac repressor protein, is also considered part of the operon although it is located at a distant site in the DNA. The i gene is constitutively expressed (not regulated) thtis, copies of the lac repressor protein are always in the cell. 

The lac repressor (encoded by the i gene), vdiidi binds to a DNA sequence called the operator... 

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