A wealth of protein and DNA sequence data is being generated by genome projects and other sequencing efforts. A crucial barrier to deciphering these sequences and understanding the relations among them is the difficulty of detecting subtle local residue patterns common to multiple sequences. Such patterns frequently reflect similar molecular structures and biological properties. A mathematical definition of this "local multiple alignment" problem suitable for full computer automation has been used to develop a new and sensitive algorithm, based on the statistical method of iterative sampling. This algorithm finds an optimized local alignment model for N sequences in N-linear time, requiring only seconds on current workstations, and allows the simultaneous detection and optimization of multiple patterns and pattern repeats. The method is illustrated as applied to helix-turn-helix proteins, lipocalins, and prenyltransferases.

/pdf/detecting-subtle-sequence-signals-a-gibbs-sampling-strategy-56bu9neqln.pdf

Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

Fused protein domains inhibit DNA binding by LexA.

http://www.jbc.org/content/265/32/19990.full.pdf

The tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product.

The structure of the 84 residue DNA binding domain of the Escherichia coli LexA repressor has been determined from NMR data using distance geometry and restrained molecular dynamics. The assignment of the 1H NMR spectrum of the molecule, derived from 2- and 3-D homonuclear experiments, is also reported. A total of 613 non-redundant distance restraints were used to give a final family of 28 structures. The structured region of the molecule consisted of residues 4-69 and yielded a r.m.s. deviation from an average of 0.9 A for backbone and 1.6 A for all heavy atoms. The structure contains three regular alpha-helices at residues 6-21 (I), 28-35 (II) and 41-52 (III), and an antiparallel beta-sheet at residues 56-58 and 66-68. Helices II and III form a variant helix-turn-helix DNA binding motif, with an unusual one residue insert at residue 38. The topology of the LexA DNA binding domain is found to be the same as for the DNA binding domains of the catabolic activator protein, human histone 5, the HNF-3/fork head protein and the Kluyveromyces lactis heat shock transcription factor.

Solution structure of the LexA repressor DNA binding domain determined by 1H NMR spectroscopy.

A Bacillus subtilis strain deficient in homologous recombination was isolated from a library of Tn917lac insertion mutants. The interrupted locus consists of an open reading frame encoding a 22,823-dalton polypeptide. Analysis of the deduced amino acid sequence revealed 34% identity and 47.3% similarity with the LexA protein from Escherichia coli. The gene was designated dinR. It is located between the recA and thyA genetic markers, at 162 degrees on the B. subtilis chromosome. The dinR gene was shown to be expressed during the entire B. subtilis cellular cycle with at least a threefold increase when cells develop competence. In addition, the use of a merodiploid strain, in which a copy of the wild-type dinR gene coexists with a dinR-lacZ transcriptional fusion, demonstrated that dinR is an SOS gene and that the SOS-induced expression of dinR occurred only when a wild-type copy of dinR was present. In addition, DinR seems to regulate the expression of dinC, another SOS gene.

Identification of dinR, a DNA damage-inducible regulator gene of Bacillus subtilis.

The amino-terminal DNA binding domain of LexA repressor consisting of 84 amino acid residues has been studied by two-dimensional 1H NMR. Sequence-specific 1H resonance assignments were made for the first 60 amino acid residues. The secondary structure of this part of the protein contains three alpha-helices in the peptide segments 8-20, 28-35, and 41-54. The last helix has a distortion around residues 47-48. The peptide segment 28-47 shows weak homology with other helix-turn-helix proteins. To investigate the spatial structure of this region of the molecule distance-geometry calculations were performed based on proton-proton distance constraints from nuclear Overhauser effects. The resulting structure shows that the segment 28-47 contains two helices with a loop region between them. The relative orientation of the two helices is similar to that found in helix-turn-helix proteins, but the helices are further apart, with the phenyl ring of Phe-37 located between them. The Brookhaven Protein Data Bank was searched for structurally homologous peptide segments in other proteins. The result of this search was that the two-helical structure of LexA is not more closely related to the canonical helix-turn-helix motif than it is to similar substructures found in other classes of proteins.

The amino-terminal domain of LexA repressor is alpha-helical but differs from canonical helix-turn-helix proteins: a two-dimensional 1H NMR study.

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The amino-terminal domain of LexA repressor is alpha-helical but differs from canonical helix-turn-helix proteins: a two-dimensional 1H NMR study.