Biomedical subjects
B Müller-Hill
Publications and source records attributed to B Müller-Hill.
How Lac repressor finds lac operator in vitro.
Filter-binding and gel mobility shift assays were used to analyse the kinetics of the interaction of Lac repressor with lac operator. A comparison of the two techniques reveals that filter-binding assays with tetrameric Lac repressor have often been misinterpreted. It has been assumed that all complexes of Lac repressor and lac operator DNA bind with equal affinity to nitrocellulose filters. This assumption is wrong. Sandwich or loop complexes where two lac operators bind to one tetrameric Lac repressor are not or are only badly retained on nitrocellulose filters under normal conditions. Taking this into account, dimeric and tetrameric Lac repressor do not show any DNA-length dependence of their association and dissociation rate constants when they bind to DNA fragments smaller than 2455 base-pairs carrying a single symmetric ideal lac operator. A ninefold increased association rate to ideal lac operator on lambda DNA is observed for tetrameric but not dimeric Lac repressor. It is presumably due to intersegment transfer involving lac operator-like sequences.
Identification of cDNAs encoding two novel rat pancreatic serine proteases.
Serine proteases (SPs) are a family of physiologically important and versatile enzymes. We designed degenerated oligodeoxyribonucleotide primers derived from the consensus amino acid aa sequences of the active site of mammalian SPs, to selectively amplify in a polymerase chain reaction (PCR) cDNA fragments coding for SPs. We used poly(A)+ RNA from rat pancreas to obtain the cDNA. Two of the amplified cDNA fragments encode novel SPs. The full-length nucleotide sequence of both cDNAs was also obtained by PCR. The high degree of homology to trypsins and elastases suggests that the cDNAs encode a trypsin-like and an elastase-like SP, respectively. Both mRNAs were also found to occur, to a lesser extent, in spleen, as was the case for the mRNAs of other rat pancreatic SPs.
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.
A model of the lac repressor-operator complex based on physical and genetic data.
Computer graphics were used to build a molecular model of the complex of Lac repressor and lac operator. The model is based (a) on the NMR data of the Kaptein group [Boelens, R., Lamerichs, R. M. J. N., Rullmann, J. A. C., van Boom, J. H. & Kaptein, R. (1988) Protein Sequence Data Anal. 1, 487-498] and (b) on our genetic and biochemical data including specificity changes [Lehming, N., Sartorius, J., Kisters-Woike, B., von Wilcken-Bergmann, B. & Müller-Hill, B. (1990) EMBO J. 9, 615-621]. Effects of amino acid exchanges in the recognition helix could be predicted by the model and were subsequently tested and confirmed by genetic experiments. Comparison of the modelled lac complex with the known crystallographic structures of several helix-turn-helix DNA complexes reveals striking similarities and suggests rules which govern the recognition between particular amino acid side chains and particular base pairs in these systems.
Funding of molecular biology.
The way that molecular biology is funded in Germany is damaging the universities. Germany policy makers should follow the advice of Wilhelm von Humboldt, and concentrate teaching and research in the universities.
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.
Cloning and characterization of the MboII restriction-modification system.
The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.
Linker mutagenesis in the lacZ gene of Escherichia coli yields variants of active beta-galactosidase.
Synthetic octameric oligonucleotides that code for a unique restriction site were cloned into a randomly linearized plasmid that carries the lacZ gene. The insertions were mapped by digestion with appropriate restriction endonucleases. 12 mutants were identified which carry an insertion within the lacZ gene and still express active beta-galactosidase. Small deletions or duplications of the wild-type sequence occurred at these positions which restore the correct reading frame. The insertions occurred in the first and the last third of the internal duplication of the lacZ gene and within the domain homologous to dihydrofolate reductase.
Dimer-to-tetramer assembly of Lac repressor involves a leucine heptad repeat.
The C-terminus of Lac repressor is responsible for the formation of repressor tetramers from active dimers. If properly grafted, the C-terminus of Lac repressor (amino acids 331 to 360) converts Gal repressor dimers into tetramers. Amino acids 342 to 356 of Lac repressor contain a 4-3 hydrophobic repeat of four leucines and one valine. Systematic amino acid replacements of all residues in this region show that the protein-protein interaction between repressor dimers depends mainly on the hydrophobic residues of the 4-3 repeat, which is constitutive for coiled coils. Thus the tetramerization site of Lac repressor resembles the leucine zipper motif found in a family of eukaryotic transcription factors.
lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators.
We have analyzed protein-DNA complexes formed between lac repressor and linear or differently supercoiled lac DNA (802 or 816 base-pairs in length), which carry all three natural lac operators (O1, O2 and O3) in their wild-type sequence context and spacing and compared them with constructs that contain specifically mutated "pseudo-operators" O2 or O3. We used gel retardation assays to identify the nature of the complexes according to their characteristic electrophoretic mobility and dissociation rate measurements to determine their stability. With linear DNA we found only indirect evidence for loop formation between O1 and O2. In covalently closed DNA minicircles the formation of a loop between O1 and O2 could be demonstrated by the observation that O1-O2 containing DNA with low negative supercoiling (sigma = -0.013 and less) is constricted by binding of lac repressor, resulting in an increased electrophoretic mobility. At elevated negative supercoiling (sigma = -0.025, -0.037, -0.05) O1-O2 containing DNA complexed with lac repressor migrates significantly slower than the corresponding O1-DNA, indicating loop formation. The dissociation of lac repressor-operator complexes is decreased with increasing negative supercoiling for all tested operator combinations of O1, O2 and O3. However, in the presence of at least two natural lac operators on the same DNA minicircle the enhancement of stability is particularly large. This indicates that a DNA loop is formed between these two lac operators, O1 and O2 as well as O1 and O3, since negative supercoiling is known specifically to promote the formation of looped structures. Additionally, we observe a dependence of dissociation rate on the spatial alignment of the operators as a result of changing helical periodicity in differently supercoiled DNA and consider this to be further evidence for loop formation between O1 and O2 as well as O1 and O3.
Differential splicing of Alzheimer's disease amyloid A4 precursor RNA in rat tissues: PreA4(695) mRNA is predominantly produced in rat and human brain.
Alzheimer's disease is characterized by filamentous depositions of amyloid A4 protein in the brain. The first precursor of A4 protein that has been described consists of 695 amino acids (PreA4(695)). Until now, three types of amyloid precursor mRNAs (PreA4(770), PreA4(751) and PreA4(695)), produced by alternative splicing, have been detected. We analysed the differential expression of these mRNAs in various rat tissues by PCR and show that (1) there exists a fourth type of mRNA, PreA4(714); (2) in all tissues except the brain the PreA4(695) mRNA is less abundant than the other types of mRNAs; in the brain, however, the PreA4(695) mRNA predominates by far. The same observations hold true for human tissues. The possible function of this differential splicing is discussed.
The isolation of the lac repressor.
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How Trp repressor binds to its operator.
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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.
The three operators of the lac operon cooperate in repression.
We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one 'pseudo-operator' O2 or O3 decreases repression by wild-type tetrameric Lac repressor approximately 2- to 3-fold; absence of both 'pseudo-operators' decreases repression greater than 50-fold. O1 alone represses under these conditions only approximately 20-fold. Dimeric active Lac repressor (iadi) represses the wild-type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.
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.
The sequence of a third member of the heat shock protein family in Plasmodium falciparum.
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