Ponzy Lu

Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

Genetic studies of the lac repressor. IX. Generation of altered proteins by the suppression of nonsence mutations.

Abstract We have generated more than 300 altered lac repressor proteins carrying known amino acid replacements, by employing nonsense mutations at 90 positions in the lacI gene together with eight different nonsense suppressors. This allows the substitution of lysine, serine, tyrosine, leucine and glutamine at virtually all of the respective positions in the repressor, and tryptophan at ten positions in the repressor. Since most of the nonsense sites have been correlated with specific codons in the lacI messenger RNA, in almost all cases the position of the substituted residue is known. This process results in the creation of a large number of mutant phenotypes. We have analyzed the effects of each substitution in vivo , and in several cases studied partially purified repressors in vitro . The properties of the altered proteins have been compared with the position and nature of each exchanged residue. We discuss the implications of these findings with regard to repressor structure in particular, and to protein structure in general. Further applications of the suppression method are also considered.

LAC REPRESSOR GENETIC MAP IN REAL SPACE

Here, we present a graphic display of the phenotypes of more than 4000 single amino acid substitution mutations on the three-dimensional structure of the lac repressor tetramer bound to DNA. The genetic data and the X-ray diffraction studies contribute to define an allosteric mechanism and yield a visual demonstration of the importance of core or buried residues in protein structure.

Residual dipolar couplings in nucleic acid structure determination.

Solution NMR spectroscopy of nucleic acids has been limited by the short-range nature of the nuclear Overhauser effect and scalar coupling restraints normally used in structure determination. The addition of residual dipolar couplings, obtained from slightly oriented mixtures, provides bond vector angles relative to a universal alignment tensor. The accurate determination of helix curvature, domain orientation and the stoichiometry of homomultimeric nucleic acid complexes is now possible.

.lambda. cro Repressor complex with OR3 DNA: nitrogen-15 NMR observations

15N NMR studies of the coliphage lambda cro repressor are presented. The protein has been uniformally labeled with 15N, and individual amino acids have been incorporated. Although the four C-terminal residues (63-66) were not located in the original crystallographic studies of the protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y., & Matthews, B.W. (1981) Nature (London) 290, 754], it has been proposed that the C-terminus is involved in DNA binding [Ohlendorf, D.H., Anderson, W.F., Fisher, R.G., Takeda, Y., & Matthews, B.W. (1982) Nature (London) 298, 718]. These experiments give direct verification of that proposal. [15N]Amide resonances are assigned for residues 56, 62, 63, and 66 in the C-terminus by enzymatic digestion and by 13C-15N double-labeling experiments. 15N[1H] nuclear Overhauser effects show that the C-terminus is mobile on a nanosecond time scale. Exchange experiments using distortionless enhancement via polarization transfer, which is sensitive to proton exchange on the 1/JNH (10 ms) time scale, indicate that the amide protons in the C-terminus are freely accessible to solvent. It is thus a flexible arm in solution. The binding of both specific operator and nonspecific DNA is shown to reduce both the mobility and the degree of solvent exposure of this arm. Two-dimensional 15N-1H correlation experiments using 15N-labeled cro reveal inconsistencies with previously reported 1H NMR assignments for the lysine amides [Weber, P.L., Wemmer, D.E., & Reid, B.R. (1985) Biochemistry 24, 4553]. This result suggests that those assignments require reexamination, illustrating the utility of 15N labeling for obtaining 1H resonance assignments of biomolecules. Furthermore, isomerization of the peptide bond of Pro-59, which has been previously suggested (Weber et al., 1985) and which would significantly affect the properties of the C-terminal arm, is shown to not occur.

λ cro Represser complex with OR3 operator DNA: 19F nuclear magnetic resonance observations

Abstract The interaction of λ cro represser with DNA is probed using synthetic 17 base-pair O R 3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue. By monitoring changes in chemical shift of the fluorine resonances upon cro represser binding in aqueous buffers of varying 2 H 2 O content, we have examined the specific cro repressor-O R 3 DNA complex in detail. The results are interpreted in the context of the popular model for cro repressor-O R 3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA. The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model.

Possible molecular detent in the DNA structure at regulatory sequences.

A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.

Lac repressor-operator complex.

For many years the lac operon of Escherichia coli has been the paradigm for gene regulation. Recently, the structures of the lac repressor core bound to isopropyl-beta-D-1-thiogalactoside (IPTG), the intact apo lac repressor, the intact lac repressor complexes with IPTG and a 21-base-pair symmetric operator, and the refined headpiece of the repressor have been determined. These structures have provided a framework for understanding a wealth of biochemical and genetic information. An analysis of these structures, as well as a description of their function and a comparison to homologous proteins, is now possible.

[23] Studies of nucleic acids and their protein interactions by 19F NMR

Publisher Summary This chapter discusses the studies of nucleic acids and their protein interactions, by 19 F nuclear magnetic resonance (NMR). Application of 19 F NMR spectroscopy for macromolecular complexes is most useful in instances where co-crystal structures are unavailable and where the large size or the complex nature of intermolecular associations severely restricts the application of 1 H NMR. Dissection of protein-nucleic acid complexes, by standard 1H NMR methods, has been substantially hampered by overlapping resonances and is only successful when restricted to small proteins (subunit size of about 12 kD) and short or symmetric nucleic acid targets (about 20 base pairs). One would like to extend NMR to extract structural information from larger complexes, with simplified spectra, limited to a preselected local neighborhood of a probe site. The density, distribution, and symmetry of the orbital electrons about a given nucleus determine the sensitivity and the orientation dependence of the resulting chemical shift. A large, intrinsic sensitivity of the covalently bound fluorine atom stems from the anisotropic distribution of the electrons in the three 2-p orbitals. The fluorine nucleus sensitivity to its micro-environment is, therefore, large and highly orientation dependent. The observed chemical shift change, induced by a new chemical environment, is a linear combination of shielding contributions from: (a) van der Waals interactions, (b) ring current shielding, (c) electronically anisotropic groups, (d) dipoles, and (e) fluorine hydrogen bonding.

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator☆

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.