Brigitte Kisters-Woike

Mutant lac repressors with new specificities hint at rules for protein--DNA recognition.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor‐operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.

How Trp repressor binds to its operator.

We propose that the generally accepted model of a single Trp repressor dimer binding to a center of symmetry in the natural trp operator (Otwinowski et al., 1988) is wrong. We show here that the Trp repressor binds to a sequence whose center is located four base pairs either to the right or to the left of the central axis of symmetry that was previously identified. We show that: (i) the oligonucleotide used by Otwinowski et al. is not retarded by the Trp repressor in a mobility shift assay under conditions wherein a shorter oligonucleotide carrying our consensus sequence is retarded, (ii) that methylation protection experiments on the full natural operator sequence and the short oligonucleotide protect similar patterns and (iii) that by varying every base in the shorter oligonucleotide, we can demonstrate an optimal sequence for Trp repressor binding.

Cre Mutants with Altered DNA Binding Properties

The recombinase Cre of bacteriophage P1 is a member of the family of site-specific recombinases and integrases that catalyze inter- and intramolecular DNA rearrangements. To understand how this protein specifically recognizes its target sequence, we constructed Cre mutants with amino acid substitutions in different positions of the presumptive DNA binding region. Here we present the results of in vitro DNA binding and in vivorecombination experiments with these Cre mutants. Most substitutions of presumptive DNA-binding amino acids in in vitro tests resulted either in the loss of target binding or in a broadening of target recognition specificity. Of the mutations resulting in a broadening of target specificity, one, N317A, results in a reduced recombination efficiency with the wild-type loxP target but recombines, in contrast to wild-type Cre, in in vivoexperiments, with a symmetric variant of the wild-type target sequence. This target variant differs from wild-type loxP by the symmetric C to A replacement in position 6 of the inverted repeats. We propose a common multihelical DNA binding motif for the family of integrases and recombinases. This model implies a major structural rearrangement for the DNA binding region of λ integrase, analogous to the structural rearrangements of the DNA binding motifs of other proteins when contacting their target DNA.

The mouse Enhancer trap locus 1 (Etl-1): a novel mammalian gene related to Drosophila and yeast transcriptional regulator genes

A novel mouse gene, Enhancer trap locus 1 (Etl-1), was identified in close proximity to a lacZ enhancer trap integration in the mouse genome showing a specific beta-galactosidase staining pattern during development. In situ analysis revealed a widespread but not ubiquitous expression of Etl-1 throughout development with particularly high levels in the central nervous system and epithelial cells. The amino acid sequence of the Etl-1 protein deduced from the cDNA shows strong similarity, over a stretch of 500 amino acids, to the Drosophila brahma protein involved in the regulation of homeotic genes and to the yeast transcriptional activator protein SNF2/SWI2 as well as to the RAD54 protein and the recently described helicase-related yeast proteins STH1 and MOT1. Etl-1 is the first mammalian member of this group of proteins that are implicated in gene regulation and/or influencing chromatin structure. The homology to the regulatory proteins SNF2/SWI2 and brahma and the expression pattern during embryogenesis suggest that Etl-1 protein might be involved in gene regulating pathways during mouse development.

The roles of residues 5 and 9 of the recognition helix of Lac repressor in lac operator binding

We constructed expression libraries for Lac repressor mutants with amino acid exchanges in positions 1, 2, 5 and 9 of the recognition helix. We then analysed the interactions of residues 5 and 9 with operator variants bearing single or multiple symmetric base-pair exchanges in positions 3, 4 and 5 of the ideal fully symmetric lac operator. We isolated 37 independent Lac repressor mutants with five different amino acids in position 5 of the recognition helix that exhibit a strong preference for particular residues in position 2 and, to a lesser extent, in position 1 of the recognition helix. Our results suggest that residue 5 of the recognition helix (serine 21) contributes to the specific recognition of base-pair 4 of the lac operator. They further suggest that residue 9 of the recognition helix (asparagine 25) interacts non-specifically with a phosphate of the DNA backbone, possibly between base-pairs 2 and 3.

On the conservation of protein sequences in evolution

The amino acid sequences of enzymes like alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase are strongly conserved across all phyla. We suggest that the amino acid conservation of such enzymes might be a result of the fact that they function as part of a multi-enzyme complex. The specific interactions between the proteins involved would hinder evolutionary change of their surfaces.

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.

Macromolecular interactions: tracing the roots: Response from Kisters-Woike et al.

We are grateful to James Clegg and his colleagues for pointing out to the readers of TiBS that a large number of papers have been published on an issue that we raised in our article, namely macromolecular interactions. ‘Alles Gescheidte ist schon gedacht worden, man muss nur versuchen es noch einmal zu denken’ (everything clever has already been thought, one must just try to think it once more), wrote Goethe 1xSee all References1. He is certainly right. The way we looked at the matter seemed novel to us. It is up to the readers of TiBS to decide whether it is also novel for them.

Mutant bZip-DNA complexes with four quasi-identical protein-DNA interfaces.

The complex between the yeast transcriptional activator GCN4 and the palindromic ATF/CREB site 5′‐ A4T3G2A1C0*G0′T1′C2′A3′T4′‐3′ shows dyad symmetry. The basic region of GCN4 contains a segment of 18 amino acids with a partially palindromic sequence: N‐LKRARNTEA*ARRSRARKL‐C. Symmetric residues are underlined. Apart from the ATF/CREB site, GCN4 also binds well to the symmetric variants with guanine in position 4 (5′‐G4T3G2A1C0*G0′T1′C2′A3′C4′‐3′) or thymine in position 0 (5′‐A4T3G2A1T0*A0′T1′C2′A3′T4′‐3′). The half‐sites of these sequences can be regarded as short pseudo‐palindromes with central guanine 2/cytosine 2′ base pairs. We investigated whether the geometry of the peptide of the basic region of GCN4 could be functionally related to the pseudo‐palindromic character of some target half‐sites. Since inspection of the X‐ray structures of GCN4‐DNA complexes reveals that several amino acid‐DNA interactions are symmetric within the wild‐type half‐complexes, we introduced mutations into a GCN4 bZip peptide that improve the symmetry of the peptide. We found that most of the constructs retain specific DNA recognition. For one mutant, we conclude that it is not only capable of forming DNA complexes showing the well‐known overall dyad symmetry, but that the protein‐DNA interface of each half‐complex can be divided further into two quasi‐identical, quasi‐symmetric substructures.