N-terminal sequence of phage lambda repressor (original) (raw)
Related papers
Biochemistry, 1977
The stoichiometries of binding of non-operator DNA and inducer to lac repressor, as well as some conformational aspects of these interactions, are described in this paper. It is shown that the circular dichroism (CD) spectrum of the repressor-non-operator DNA complex is appreciably different from that obtained by summing the spectra of the separate components; the major change is a substantial enhancement of the positive (-275 nm) lobe of the originally conservative DNA B form pattern. These CD spectral changes appear to reflect a change in DNA conformation on repressor binding and are interpreted in terms of tilting (relative to the DNA axis) of some of the base pairs of the native structure, or perhaps some twisting of the overall structure resulting in a tighter coupling of vicinal base transition moments. These changes in the CD spectrum of non-operator DNA on repressor binding have been monitored as a function of added repressor concentration, under tight-binding conditions, to establish that the site size (n) for binding to non-operator DNA is -12 base pairs per repressor tetramer (-24 base pairs if repressor binds to both sides of the double helical DNA lattice). This value of n is confirmed both by calculation from binding isotherms , Biochemistry 16 (following paper in this issue), and by titration of repressor T h e lac repressor recognizes (and binds tightly to) a particular sequence of base pairs in the Escherichia coli chromosome which is defined, on this basis, as the lac operator. By virtue of this specific binding the repressor exerts negative control over the expression of the lactose operon (Jacob and . The interaction of lac repressor with operator has been subjected to intense scrutiny by both biochemical and genetic means, and a great deal of information is now available State University, East Lansing, Mich. 48824. sulfhydryl groups as a function of added DNA concentration. The measured site size is discussed in terms of the relationship between operator and non-operator DNA binding of repressor, and of various features of the known operator sequence, to suggest alternative models for the geometry of the repressoroperator interaction. Investigation of the repressor-inducer interaction by equilibrium dialysis, fluorescence, and gel permeation chromatography shows, in confirmation of the results of Ohshima, Y., et al. , that different repressor preparations exhibit different (average) numbers of "active" inducer binding sites per repressor tetramer (nl); values of nl between two and four have been obtained. The effects on n~ of a variety of environmental conditions have been examined, and the results, together with relevant data from the literature, are discussed in terms of conformational equilibria between forms of repressor subunits which bind strongly to inducer and weakly to operator (RI), and forms which bind strongly to operator and weakly to inducer (Ro). Thermodynamic parameters for the binding of inducer to repressor subunits (in the RI form) have also been determined. At pH 7.6 (4-25 "C), AGO = -7.7 kcal/mol (25 "C), AH" = -6.2 kcal/mol, and AS" = +5 cal mol-' deg-' for this reaction. about both the protein and the operator (for a recent review, see .
Completed DNA sequences and organization of repressor-binding sites in the operators of phage lambda
Journal of Molecular Biology, 1977
We complete the determination of the DNA sequences of the bacteriophage ~t operators by presenting two new sequences, one about 38 base-pairs long from the left operator (0~), and another about 42 base-pairs long from the right operator (OR). These were determined by direct DNA sequencing. The results of experiments in which each operator was digested with nuclease in the presence of highly purified ~ repressor are also described. From the new sequence information, as well as other considerations, including the results of the nuclease digestion experiments, we conclude that each operator is smaller than previously reported and consists of three repressor-binding sites. The sequence near OR includes part of the repressor structural gene, cI.
Journal of Molecular Biology, 1996
Each amino acid from position 2 to 329 of Lac repressor was replaced by 1 Institut for Genetics 12 or 13 of the 20 natural occurring amino acids. The resulting phenotypes University of Cologne are discussed on the basis of (1) the recently published structure of the Lac Weyertal 121, 50931 Cologne, Germany repressor core complexed with the inducer IPTG and (2) a model of the dimeric Lac repressor built by homology modelling from the X-ray 2 Molecular Biology Institute structure of the purine repressor-corepressor-operator complex. This and Department of Biology phenotype analysis, based on 4000 well-defined mutants, yields a University of California functional description of each amino acid position of Lac repressor. In most Los Angeles, CA 90024 cases, mutant effects can be directly correlated with the structure and USA 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 3 Plum Island Animal Disease not complicated: amino acids which are directly involved in sugar binding Center, P.O. Box 848 are affected in Lac repressor mutants of the I S type; small amino acids Greenport, NY 11944, USA 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.
Biochemistry, 2008
Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI: Upon DNA release, LacI shows a 20Å increase in length that was previously attributed to unfolding the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally-occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants show altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.
GENETIC ANALYSIS OF THE ACTIVE SITES OF lac REPRESSOR
Genetics, 1974
Selection methods for the isolation of i -d and i s mutants are described. Two hundred and forty-seven i -d and 98 i s mutations have been localized by deletion mapping. All i -d mutations map in the region of the i gene, which codes for the aminoterminal part of the lac repressor, whereas i s mutations map in the middle of the i gene and at the proximal end of the i -d cluster.
Lac repressor genetic map in real space
Trends in Biochemical Sciences, 1997
Here, we present a graphic display of the phenotypes of more than 4000 single amino acid substitution mutations on the three-dimensional structure of the lac repressor tetramer bound to DNA. The genetic data and the X-ray diffraction studies contribute to define an allosteric mech.nism and yield a visual demonstration of the importance of core or buried residues in protein structure.
Repressor-operator interaction in the lac operon
Journal of Molecular Biology, 1982
We have examined the N-terminal 56 amino acid fragment, the domain that can bind DNA independently, from 3-fluorotyrosinr-substituted Escherichin coli Zac repressor by "F-nuclear magnetic resonance. The fragments or "headpieces" from four altered repressors missing each of the tyrosines in turn were examined in parallel. When the wild-type N-terminal fragment is titrated with a 36 base-pair Inc operator DNA sequence? the "F resonances undergo changes in their chemical shifts that are different from those changes when the N-terminal fragment is Gtrated wit.h non-specific DNA fragments. By looking at these operator-induced changes as well as pH-dependent effects with all four altered N-terminal fragments, we show systematic correlations with the genetic data. The data lead us to conclude that upon operator DNA binding: (1) tyrosine 7 is displaced to a less polar environment and the higher than normal pK value of the phenolic OH group is decreased; (2) tyrosine 12 does not change much in either its mobility or environment; and (3) tyrosine 17 is involved. as suggested by the genetir data. when the headpiece forms a complex with operator DSA. lrrc REPRESSOR OPERATOR ISTERA('TlC)NS. ITT 453 c*
Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors
Journal of Molecular Biology, 1997
The wild type E. coli lac operator is embedded in a 35 base-pair DNA 1 Department of Chemistry sequence containing extensive 2-fold symmetry, suggesting a symmetric University of Pennsylvania repressor operator complex. However, deviations from strict 2-fold Philadelphia PA 19104, USA symmetry occur at the central base-pair and at three additional base-pairs. 2 Department of Biochemistry