Genetic studies of the lac repressor

Miller, Jeffrey H; Coulondre, Christine; Hofer, Murielle; Schmeissner, Ursula; Sommer, Hans; Schmitz, A.; Lu, Ponzy

doi:10.1016/0022-2836(79)90073-1

Cited by 128 publications

(46 citation statements)

References 35 publications

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“…For mutant isolation E. coli RR1 (pro leu thi lacZ gal xyl ara mtl hsdS phx supE rpsL) or its recA13 derivative HB101 was used. Growth of X421 was on strain JMsu6 [A(lac pro) ara argE(Am) nalA rifthi supB(Am)] (19). For coconjugation experiments we used as a donor strain PK191 [Hfr thi A(lac pro) phx sup (Am) colV], a noncolicinogenic derivative of PK19 (11).…”

Section: Methodsmentioning

confidence: 99%

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

1988

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A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.

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Section: Methodsmentioning

confidence: 99%

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

1988

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“…Therefore, in this study, we analyzed 83 of the 557 residues in KdpA to identify those that are critical for substrate binding. To test comprehensively the importance of each residue, we examined at least 11 substitutions at each residue using the amber suppression technique (8,9). This technique creates up to 12 amino acid substitutions at an introduced amber stop codon.…”

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confidence: 99%

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Dorus¹,

Mimura²,

Epstein

2001

Journal of Biological Chemistry

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The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K ؉ with high affinity and specificity. ؉ binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K ؉ binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K ؉ affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K ؉ , an indication that a residue is in or very near a K ؉ binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K ؉ and thus reflects the effective distance between a residue and the site of binding or passage of K ؉ in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K ؉ as it moves through the KdpA subunit to cross the membrane.

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“…To obtain enough operator DNA, it was polymerized in tandem and cloned in a plasmid (22). Second, our ability to describe and assign the specific changes in the NMR spectrum of the repressor-lac operator complex comes from a comprehensive genetic study ofthe protein (23,24).…”

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confidence: 99%

lac repressor-lac operator interaction: NMR observations.

Nick¹,

Arndt²,

Boschelli³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues-four tyrosines and a single histidine-in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by '9F NMR when compared with 1H NMR of otherwise identical repressor-DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.The lac repressor-lac operator interaction is the prototype system in revealing the molecular mechanism involved in gene regulation (1)(2)(3). Extensive and detailed information has been accumulated about the DNA sequence requirements and some of the important features of the individual bases in the DNA sequences necessary for proper lac repressor-lac operator DNA complex formation (4-7). Equivalently detailed information about the involvement of specific sites on the lac repressor protein is lacking. It is normally a tetrameric protein made up of four identical subunits with known sequence of360 amino acids each (8). Limited chymotrypsin digestion of the protein gives a tetrameric core containing amino acid residues 57-360 and four amino-terminal peptides containing residues 1-56 (9). The amino-terminal domain, or headpiece, which appears to be part of the DNA binding site, contains five aromatic residues: four tyrosines and a histidine (9-12). We show here changes in the NMR signals from these residues when it is titrated with a 36-base-pair (bp) double helical DNA fragment containing the lac operator sequence.We exploit the advantages of using lac repressors containing the nuclear spin label, 19F, attached to the 3 position on the tyrosine rings. The substituted repressor binds operator DNA and releases it upon binding the inducer (13,14).Direct spectroscopic observations of lac repressor-nonspecific DNA interactions have been made by using absorption, fluorescence, and circular dichroism (15)(16)(17)(18). Similar methods (19) and NMR (20,21) have been used to look at headpiece nonspecific DNA interaction. The experiments that are described here represent the combination of two systematic efforts to understand the molecular nature of the specific protein-DNA interaction. First, the operator DNA fragment was synthesized as part of a program to explore the recognition sites on the lac operator DNA sequence (6, 7). To obtain enough operator DNA, it was polymerized in tandem and cloned in a plasmid (22). Second, our ability to describe and assign the specific changes in the NMR spectrum of the repressor-lac operator complex comes from a comprehensive genetic study ofthe protein (2...

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Genetic studies of the lac repressor

Cited by 128 publications

References 35 publications

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

lac repressor-lac operator interaction: NMR observations.

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