Operator DNA

The consequences of these changes were impressive. The parental 434 repressor had no affinity for the P22 operator in vivo, but the redesigned 434 repressor controiled only P22 operators and not 434 operator regions in vivo. Purified redesigned 434 repressor bound specifically to P22 operator DNA in vitro and showed the same hierarchy of affinities for the three regions ORl, 0R2, and OR3 as native P22 repressor. 

The elegant genetic studies by the group of Charles Yanofsky at Stanford University, conducted before the crystal structure was known, confirm this mechanism. The side chain of Ala 77, which is in the loop region of the helix-turn-helix motif, faces the cavity where tryptophan binds. When this side chain is replaced by the bulkier side chain of Val, the mutant repressor does not require tryptophan to be able to bind specifically to the operator DNA. The presence of a bulkier valine side chain at position 77 maintains the heads in an active conformation even in the absence of bound tryptophan. The crystal structure of this mutant repressor, in the absence of tryptophan, is basically the same as that of the wild-type repressor with tryptophan. This is an excellent example of how ligand-induced conformational changes can be mimicked by amino acid substitutions in the protein. 

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 product of the repressor gene, the 236-amino-acid, 27 IcDa repressor protein, exists as a two-domain molecule in which the amino terminal domain binds to operator DNA and the carboxyl terminal domain promotes the association of one repressor protein with another to form a dimer. A dimer of repressor molecules binds to operator DNA much more tighdy than does the monomeric form (Figure 39-6A to 39-6C). 

The product of the cro gene, the 66-amino-acid, 9 kDa Cro protein, has a single domain but also binds the operator DNA more tightly as a dimer (Figure... 

Cocrystals of Escherichia coli Trp repressor bound to an alternative operator DNA sequence. J. Mol. Biol. 234,496-498. 

Nelson, H. C. and Sauer, R. T. (1986). Interaction of mutant lambda repressors with operator and non-operator DNA. /. Mol. Biol. 192,27-38. 

Pace, H. C., Lu, P and Lewis, M. (1990). lac repressor crystalhzation of intact tetramer and its complexes with inducer and operator DNA. Proc. Natl. Acad. Sci. USA 87,1870-1873. 

Catabolite repression is a two-part system. The first component is the small-molecule regulator, cyclic AMP. Glucose decreases cyclic AMP synthesis. The second component is cyclic AMP binding protein, CAP. CAP binds cAMP and thereby helps RNA polymerase bind to the promoter. When bound to cAMP, CAP binds to a sequence at the 5 end of the lac promoter. CAP binding bends the DNA, allowing protein-protein contact between CAP and polymerase. It therefore behaves in the opposite manner of repressor. Repressor (LacI) binds to operator DNA only in the absence of its small-molecule ligand, while CAP binds to promoter DNA in the presence of its small-molecule ligand. 

Several crystal structure analyses of individual repressors and of repressor-operator complexes disclosed a recurrent template on the protein side. It consists of an a-helix/turn/a-helix motif with an almost invariant glycine in the turn region [707-710]. Most of the repressors so far investigated display such a motif but the interactions with the operator DNA, which occur in the major groove are individually different. Here we consider more closely the well-documented complexes between the N-terminal fragment of the repressor from phage 434 and its specific operator DNA [711], and the ternary complex formed between tryptophan repressor, tryptophan and DNA [712, 713]. 

In the crystal structure [711], a dimes formed by two N-terminal 434 repressor fragments is bound to the 20 base pairs DNA duplex so that the complex has overall 2-fold rotational symmetry. The polypeptide chain is folded into five a-helices HI to H5, with helices H2 and H3 forming the helix/turn/helix motif (Fig. 20.16). Helices H3 and H3 of the 434 repressor dimer insert into two successive major grooves of the operator DNA whereas the N-termini of the flanking helices H2, H4 and H2 H4 contact the sugar-phosphate backbones. 

Fig. 20.16. Stereo view of the phosphate backbone of the 20 base-pairs long operator DNA fragment in complex with the a-carbon backbone of the dimeric 434 repressor fragment. The dashed lines connect phosphates in the minor groove, which is compressed in the center. HI to HS denote helices 1 to 5, arrows point at close protein-DNA contacts (from [711])...

Fig. 20.17. Stereo view showing the specific recognition between 434 repressor fragment and operator DNA. Dotted lines are hydrogen bonds with X - A distances <3.5 A, open circles are water molecules (from [711])...

Conformational changes of trp (tryptophane) repressor and water-mediated contacts to DNA. trp Repressor is a 108 amino acids long polypeptide folded into 6 a-helices A to F. It was investigated by high resolution X-ray diffraction studies as apo repressor, as binary complex with its corepressor tryptophan, and as ternary complex bound to its specific operator DNA, a palindromic self-complementary 18-mer with overhanging T [712, 713] ... 

Fig. 20.20. Stereodiagram showing how the structure of the trp aporepressor (shaded cylinders) changes after binding to tryptophan and to operator DNA (open cylinders). From [712]...

A second demonstration of participation in renaturation of heat-inactivated proteins by dnaK is provided by experiments with a temperature-sensitive repressor protein of bacteriophage X, XcI857 protein (Gaitanaris et ai, 1990). Activities of the Xcl wild-type and mutant proteins were measured in an in vitro operator DNA binding assay and by in vivo expression from a Xcl-regulated operon fusion of XPrOr and the... 

For NMR samples the protein was concentrated by ultrafiltration to a final concentration of 1-2.4 mM trp repressor monomer in 500 mM NaCl, 50 mM sodium phosphate, pH6. The high salt concentration was necessary to prevent aggregation of die protein. The corepressors, either L-tryptophan or 5-methyl-L-tryptophan, were added at a concentration of 1.5-2 times the protein subunit concentration to form trp holorepressor. Protein-DNA complexes were prepared by adding the appropriate amount of synthetic operator DNA (23) to the above sample followed by dialysis and concentration into a pH6 solution of 50 mM (or less) sodium phosphate. Higher salt concentrations destabilized the protein-DNA complex and resulted in much poorer quality NMR spectra. 

Figure 31.6. LAC Repressor-DNA Interactions. The lac repressor DNA-binding domain inserts an a helix into the major groove of operator DNA. A specific contact between an arginine residue of the repressor and a G-C base pair is shown at the right.

Nucleic Acid Model Building Multiple Conformations of E. coli tip Operator DNA. ... 

When the complexes between the lactose operator and operator DNA [22] or that between the repair protein and abasic site-bearing DNA [23] are irradiated, the complexes are destroyed mainly due to the damage to the protein. When irradiated alone the lactose repressor or the repair protein Fpg lose their ability to bind DNA at a dose that is much lower than that necessary for destroying the complexes. The explanation lies in the reverse radioprotection provided to the protein by the DNA. RADACK calculations using the structural parameters for a free and a complexed repressor headpiece (Fig. 5) reveals the most probable sites of protein lesions. 

How does the lac repressor locate the operator site in the F. coli chromosome The lac repressor binds 4 X 10 times as strongly to operator DNA as it does to random sites in the genome. This high degree of selectivity... 

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