A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene.
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Bacterial Proteins↗
Biomedical subjects
M Ptashne
Publications and source records attributed to M Ptashne.
A phage repressor-operator complex at 7 A resolution.
The crystal structure of a complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator shows that the protein, very similar in conformation to gamma repressor, binds to B-form DNA with the second alpha-helix of a helix-turn-helix motif lying in the major groove.
Bacteriophages↗
Changing the binding specificity of a repressor by redesigning an alpha-helix.
We replaced amino acids on the 'outside', or solvent-exposed, surface of the DNA recognition alpha-helix of 434 repressor with the corresponding amino acids from the recognition helix of P22 repressor. The binding specificity of the resulting hybrid protein, as measured in vivo and in vitro, was that of P22 repressor.
Amino Acid Sequence↗
Ethylation interference and X-ray crystallography identify similar interactions between 434 repressor and operator.
In the crystal structure of a repressor-operator complex (the 434 repressor DNA-binding domain and its 14-base pair (bp) operator), Anderson et al. elsewhere in this issue identify six positions of likely contact between repressor protein and phosphates of the DNA backbone. At each of these positions, electron densities of protein and DNA merge. Experiments presented here indicate that intact 434 repressor approaches these phosphates very closely when it is bound to DNA in solution. Specifically, when any one of these phosphates is ethylated, repressor cannot bind to the modified operator. We also identify another position where ethylation has a significant but less dramatic effect on repressor binding, and note that in the structure, repressor closely approaches this phosphate. Our results strongly support the idea that the interactions between protein and the DNA phosphate backbone in the crystallized complex are the same as those made by intact repressor to operator DNA in solution. In addition, our results suggest that DNA is slightly bent by repressor binding.
Alkylation↗
Gene regulation by proteins acting nearby and at a distance.
Transcription of genes can be controlled by regulatory proteins that bind to sites on the DNA either nearby or at a considerable distance. Recent experiments suggest a unified view of these apparently disparate types of gene regulation.
DNA Helicases↗
DNA loops induced by cooperative binding of lambda repressor.
It has been shown by Hochschild and Ptashne that lambda repressors bind cooperatively to operator sites separated by five or six turns of the helix. Cooperative binding is not observed if the sites are separated by a nonintegral number of turns, unless a four-nucleotide gap is introduced into one of the strands between the two sites. These and other facts suggested that repressors at the separated sites touch each other, the DNA bending smoothly so as to accommodate the protein-protein interaction. Here we use electron microscopy to visualize the predicted protein-DNA complexes.
Binding Sites↗
Structure of the repressor-operator complex of bacteriophage 434.
The crystal structure of a specific complex between the DNA-binding domain of phage 434 repressor and a synthetic 434 operator DNA shows interactions that determine sequence-dependent affinity. The repressor recognizes its operators by its complementarity to a particular DNA conformation as well as by direct interaction with base pairs in the major groove.
Base Composition↗
Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro.
The repressor of phage 434 binds to six operator sites on the phage chromosome. A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig. 1b). In contrast, the sequences of the inner four base pairs are highly variable. Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig. 1a). We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor. We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434. We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.
Base Sequence↗
A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact.
The repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the DNA. We have identified an amino acid-base pair contact that determines (in part) the DNA-binding specificity of 434 repressor. The identification is based on the properties of a 'new-specificity' mutant, named Repressor [Ala 28], which bears the substitution of Ala for Gln at the first residue of its recognition alpha-helix. Repressor [Ala 28] binds with high affinity to a particular doubly mutant operator bearing the same substitution at position 1 in each half-site, but does not bind to either the wild-type operator or to other mutant operators. We describe molecular models of residue 28-base pair 1 interactions that account for the binding specificities of both the mutant and wild-type proteins.
5-Methylcytosine↗
Transcription in yeast activated by a putative amphipathic alpha helix linked to a DNA binding unit.
Gene activation by a DNA-binding regulatory protein in yeast requires the protein to have two components: one to recognize a specific DNA sequence and a second, the 'activating region', to interact with a general transcription factor or perhaps with RNA polymerase. The activating regions that have been characterized are acidic, and mutational analysis of one indicates that this acidity is important for activity. Here we report the design of an artificial protein bearing a novel 15-amino acid peptide linked to a DNA binding fragment of the yeast regulatory protein GAL4). The synthetic peptide is acidic and should it form an alpha-helix, that helix would be amphipathic, having one hydrophilic face bearing the acidic residues, and one hydrophobic face. When expressed in yeast, the artificial protein bearing this peptide efficiently activates the GAL1 gene which is ordinarily activated by GAL4. An otherwise identical protein with the novel 15 amino acids in a scrambled order, and which is thus unable to form an amphipathic structure, does not activate GAL1 transcription.
Binding Sites↗
Repressor, operators, and promoters in bacteriophage lambda.
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Binding Sites↗