The different faces of science: is genetics a social construct?
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Eugenics↗
Biomedical subjects
B Müller-Hill
Publications and source records attributed to B Müller-Hill.
A lethal mutant of the catabolite gene activator protein CAP of Escherichia coli.
The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses its recognition helix to bind with each subunit the DNA sequence motif 5' G-7T-6G-5A-4 3'. It makes a direct amino acid-base contact with E181 and cytosine in position-5' on the reverse strand. While testing mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is lethal in high amounts for the E. coli strains we used for cloning. We cloned this CAP mutant successfully in cya- strains, where CAP is inactive. Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity when present in low, non-lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets. It binds well to CAP consensus with G or T in position-5, better to CAP consensus with A, C in position-5, quite well to lambda consensus operator with G in position-7 and rather weakly to lambda consensus.
Bacterial Proteins↗
How AraC interacts specifically with its target DNAs.
Previous work indicates that one subunit of the AraC protein dimer binds to a DNA target araI1, of 17 base-pairs. We systematically substituted every base-pair in a synthetic araI1 target with the three possible alternatives and then tested binding of araI1 and of these 51 DNA targets to AraC by quantitative gel shift analysis in the presence of L-arabinose. We found that every substitution of the underlined bases reduces AraC binding tenfold or more: 5' TAGCATTTTTATCCATA 3'. Substitutions at other bases have little or no effect. In the absence of L-arabinose we observed a sixfold reduction of binding of AraC to araI1. We have designated the 5' AGC sequence the A-box and the 5'TCCATA sequence the B-box. We synthesised DNA targets containing either two A or two B-boxes with the natural araI1-I2 spacing. Wild-type AraC binds both targets in the presence of L-arabinose in a gel shift-experiment. In the absence of L-arabinose, AraC binds only to the double B-box. We then tested various AraC mutant proteins in the same way. S208A and H212A bind to the double B-box but not to the double A-box in the presence or absence of L-arabinose. D256A binds to the double A-box, but not to the double B-box, in the presence of L-arabinose but not in its absence. The implications of these results for the mechanism of AraC induction by L-arabinose are discussed.
Amino Acid Sequence↗
Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure.
Each amino acid from position 2 to 329 of Lac repressor was replaced by 12 or 13 of the 20 natural occurring amino acids. The resulting phenotypes are discussed on the basis of (1) the recently published structure of the Lac repressor core complexed with the inducer IPTG and (2) a model of the dimeric Lac repressor built by homology modelling from the X-ray structure of the purine repressor-corepressor-operator complex. This phenotype analysis, based on 4000 well-defined mutants, yields a functional description of each amino acid position of Lac repressor. In most cases, mutant effects can be directly correlated with the structure and function of the protein. This connection between the amino acid position and the structure and function of the protein is in most cases direct and not complicated: amino acids which are directly involved in sugar binding are affected in Lac repressor mutants of the Is type; small amino acids which can only be replaced by other small acids are located in the core of the protein; positions at which nearly all amino acids are tolerated are in most cases located on the surface of the protein. Amino acids which are highly conserved throughout the LacI family of repressors, and not directly involved in specific functions of the protein like DNA recognition or sugar binding, form a network of contacts with other amino acids. Such amino acids are either located inside one subunit, mostly at the interface between secondary structure elements, or are involved in the dimerisation interface.
Amino Acid Sequence↗
Mutants in position 69 of the Trp repressor of Escherichia coli K12 with altered DNA-binding specificity.
Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.
Amino Acids↗
The side-chain of the amino acid residue in position 110 of the Lac repressor influences its allosteric equilibrium.
Binding of the Lac repressor to its operator DNA controls the expression of the genes of the lac operon of Escherichia coli. Lac repressor's affinity for the lac operator is diminished by an inducer that affects the structure of the repressor tetramer. Here we report the cloning and sequencing of the mutant Lac repressor i-t gene, whose product, the LacR-t repressor, shows a higher affinity for the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and a lower affinity for the lac operator than the wild-type repressor. We show that the altered phenotype is due to a single amino acid residue replacement; the alanine residue at position 110 in the wild-type is replaced by threonine in i-t. Other amino acid residues in position 110 have been shown to result in an i-s phenotype. For the i-s-substitution of alanine 110 with lysine we demonstrate an increase in the affinity for operator DNA and a decrease in the affinity for IPTG. Thus, A110--> K shows the opposite effect to A110-->T on the repressor protein. We explain the phenotype of the LacR mutants by displacements of the conformational equilibrium for the dimeric repressor unit between RR (high operator affinity, low inducer affinity) and R*R* (low operator affinity, high inducer affinity) towards R*R* in the i-t and towards RR in the i-s mutant in position 110 with respect to the wild-type. The putative structures of the wild-type and mutant Lac repressors confirm this conclusion.
Allosteric Regulation↗
Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator.
The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O1 and an auxiliary ideal lac operator (O(id)) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP-independent lac UV5 promoter by O1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O(id)). Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo. Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O(id) with the weaker binding lac operator O3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.
Base Sequence↗
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.
Amino Acid Sequence↗
Co-operative binding of two Trp repressor dimers to alpha- or beta-centred trp operators.
The alpha-centred trp operator binds one dimer of the Trp repressor, whereas the beta-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the alpha-centred trp operator, but it does not bind to the beta-centred trp operator. This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with alpha-centred trp operator variants showed that positions +/-1 of the alpha-centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the alpha-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T-A to G-T) effectively transform the alpha-centred trp operator into a beta-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a beta-centred trp operator.
Bacterial Proteins↗
A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.
The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.
Amino Acid Sequence↗
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↗
The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition.
We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.
Amino Acid Sequence↗
Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity.
Two residues are invariant in all bZip basic regions: asparagine -18 and arginine -10 (we define the first leucine of the leucine zipper of GCN4 as +1). X-ray structures of two specific GCN4-DNA complexes (Ellenberger et al., Cell, 71, 1223-1237, 1992; König & Richmond, J. Mol. Biol., 233, 139-154, 1993) demonstrate the involvement of both residues in specific base pair recognition. We replaced either asparagine -18 or arginine -10 with all other amino acids and tested the DNA binding properties of the resulting mutant peptides by gel mobility shift assays. Peptides with histidine -18 or tyrosine -10 bind with changed specificities to variants of the ATF/CREB site 5'A4T3G2A1C0*G0'T1'C2'A3'T4'3' with symmetric exchanges in positions 2/2' or 0/0', respectively. The double mutant with histidine -18 and tyrosine -10 combines the features of the parental single mutants and binds specifically to the respective double exchange target. Furthermore, the tyrosine -10 mutant clearly prefers the palindrome 5'ATGATATCAT3' over the corresponding pseudo-palindrome 5'ATGATTCA-T3', whereas the lysine -10 mutant binds better to the pseudo-palindromic AP1 site 5'ATGACTCAT3' than to the palindromic ATF/CREB site. Thus, although invariant within natural bZip proteins, asparagine -18 or arginine -10 can be functionally replaced by other amino acids, and their replacement can lead to new DNA binding specificities.
Amino Acid Sequence↗
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↗
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↗
Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions.
The specificity of the GCN4/DNA complex is mediated by a complicated network of interactions between the basic regions of both GCN4 monomers and their target halfsites. According to X-ray analyses (1, 2) one particular thymine of the target sequence is recognized by serine -11 and alanine -15 (we define the leucine in the first d-position of the heptad repeats as +1). We replaced serine -11 or alanine -15 with all other amino acids and analysed the DNA binding properties of the resulting stable GCN4 derivatives by electrophoretic mobility shift assays. Among these, mutants with tryptophan in position -11, or glutamic acid and glutamine in position -15, differ significantly from GCN4 in their DNA binding specificities. We then constructed selected double mutants, which differ from GCN4 in positions -11, -15 or -14 (3) of the basic region. The double mutants with tryptophan in position -11 and asparagine or serine in position -14 show drastically altered DNA binding specificities, presumably due to additive effects.
Base Sequence↗
DNA recognition and the code.
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Amino Acid Sequence↗
Cloning of the cDNA encoding human brain trypsinogen and characterization of its product.
We designed degenerated oligodeoxyribonucleotide primers derived from amino acid (aa) sequences of the highly conserved active sites of mammalian serine proteases (SPs). These primers were used to selectively amplify, in polymerase chain reactions (PCRs), cDNA fragments coding for a SP. We used poly(A)+RNA from human brain to obtain cDNA fragments and amplified one cDNA encoding a novel SP. The full-length nucleotide (nt) sequence was identified by PCR and screening a genomic library in order to obtain the 5'-region. The deduced as sequence shows a high degree of homology to trypsinogens, except for the first exon. In addition to this brain-specific trypsinogen, there exists a variant of the cDNA in pancreas, differing only in the nt sequence of the first exon. An active form of the trypsin was synthesized in vitro and purified by affinity chromatography using soybean trypsin inhibitor (STI) agarose to demonstrate the trypsin-specific interaction with a naturally occurring inhibitor of trypsins.
Amino Acid Sequence↗