Search PubMed⌕ Search

Biomedical subjects

P Kolkhof

Publications and source records attributed to P Kolkhof.

6 recordsLinked to original sources
PubMed1995

A basic tail increases repression by dimeric lac repressor.

Tetrameric Lac repressor achieves cooperative repression by binding simultaneously to O1 and to one of the auxiliary operators O2 or O3, thereby forcing the intervening DNA into a loop. Dimeric Lac repressor is not able to form DNA loops and consequently shows no cooperative repression. We constructed a dimeric Lac repressor mutant which exhibits increased repression to the lac operon that does not depend on specific operator-repressor-operator loops. This Lac repressor carries a synthetic tail of basic residues attached to its C terminus. With this construct, we observe an increase of the in vivo repression upon addition of auxiliary lac operators to a chromosomal lac operon controlled by O1. This suggests that the basic tail enables dimeric Lac repressor to enhance its repression by additional non-specific DNA contacts.

Amino Acid Sequence↗
PubMed1994

Lambda cI repressor mutants altered in transcriptional activation.

We analysed the in vivo functions of three lambda cI repressor mutants which are phenotypically defective in positive control (pc). For this purpose, we constructed a lambda cI repressor expression system which allows controlled expression of various amounts of lambda cI repressor or its mutants. A five-fold activation of the PRM promoter by wild-type lambda cI repressor is measured in this in vivo system. Two of the pc mutants (pc 1: G43-R and pc 3: E34-K) repress the PRM promoter over a wide range of intracellular concentrations, the lowest being almost identical to the concentration of wild-type lambda cI repressor at which it activates the transcription of its own gene. Only the third pc mutant (pc 2: D38-N) behaves in a manner that would be expected of a true pc mutant, which is unaffected in its DNA binding activity but has lost its activation function. We studied the DNA binding properties of cI repressor and its three pc mutants with a variety of operator constructs in vivo and found that the four repressor proteins differed significantly with respect to their affinities for all operators tested. We also probed the necessity of an acidic residue at position 38 of cI repressor for activation and found that the substitution of aspartic acid 38 by tyrosine does not reduce activation of PRM. Furthermore a substitution with phenylalanine improves the activator function of cI repressor. Our results suggest that amino acid replacements at position 34 or 43 of lambda cI repressor predominantly affect the binding properties of the repressor while some hydrophobic amino acid residues at position 38 are at least as functional in activation as the acidic wild-type amino acid residue.

Amino Acid Sequence↗
PubMed1994

Quality and position of the three lac operators of E. coli define efficiency of repression.

Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.

Base Sequence↗
PubMed1992

Specificities of three tight-binding Lac repressors.

Tight binding mutants of Lac repressor exhibit complex repression phenomena. In this work, in vivo Lac operator binding of three such mutants of E. coli Lac repressor (X86: ser 61-leu, l12: pro 3-tyr and the double mutant l12X86: pro 3-tyr, ser 61-leu) was analyzed. Repression of beta-galactosidase synthesis controlled by ideal lac operator and its 27 symmetric operator variants containing each possible base-pair at each single half-operator position in the presence of the tight-binding Lac repressor mutants was determined. The average increase of repression with all operator variants was about 3 fold with the X86 mutant. It was about 4 fold with the l12 mutant and about 2 fold with the double mutant l12X86 as compared to wildtype Lac repressor. The X86 mutant showed the same increase of affinity to all operator variants, whereas the l12 and l12X86 mutants exhibited lower repression with some variants than with most others. These results suggest that the X86 mutant has gained no additional specificity. In contrast the l12 mutant and the l12X86 mutant exhibit a relaxed specificity for certain base pairs in positions 1 and 3 of lac operator. This suggests that the extreme N-terminus of Lac repressor may interact with the inner base-pairs in the minor groove.

Amino Acid Sequence↗
PubMed1992

Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator.

Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator.

Amino Acid Sequence↗