Two helix DNA binding motif of CAP found in lac repressor and gal repressor.

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA.     

Purification and characterization of the Streptomyces lividans initiator protein DnaA.

The Streptomyces lividans DnaA protein (73 kDa) consists, like the Escherichia coli DnaA protein (52 kDa), of four domains. The larger size of the S. lividans protein is due to an additional stretch of 120 predominantly acidic amino acids within domain II. The S. lividans protein was overproduced as a His-tagged fusion protein. The purified protein (isoelectric point, 5.7) has a weak ATPase activity. By DNase I footprinting studies, each of the 17 DnaA boxes (consensus sequence, TTGTCCACA) in the S. lividans oriC region was found to be protected by the DnaA fusion protein. Purified mutant proteins carrying a deletion of the C-terminally located helix-loop-helix (HLH) motif or with amino acid substitutions in helix A (L577G) or helix B (R595A) no longer interact with DnaA boxes. A substitution of basic amino acids in the loop of the HLH motif (R587A or R589A) entailed the formation of S. lividans mutant DnaA proteins with little or no capacity for binding to DnaA boxes. Thus, like in E. coli, the C-terminally located domain IV is absolutely necessary for the specific binding of DnaA. A mutant protein lacking a stretch of acidic amino acids corresponding to domain II is not affected in its DNA binding capacity. Whether the acidic domain II interacts with accessory proteins remains to be elucidated.     

Conserved DNA binding and self-association domains of the Drosophila zeste protein.

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.     

DNA interaction and dimerization of eukaryotic SMC hinge domains

The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1alpha/SMC3 and the meiotic SMC1beta/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiled-coil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiled-coil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer.     

DNA sequence and structure: direct and indirect recognition in protein-DNA binding

MOTIVATION: Direct recognition, or direct readout, of DNA bases by a DNA-binding protein involves amino acids that interact directly with features specific to each base. Experimental evidence also shows that in many cases the protein achieves partial sequence specificity by indirect recognition, i.e., by recognizing structural properties of the DNA. (1) Could threading a DNA sequence onto a crystal structure of bound DNA help explain the indirect recognition component of sequence specificity? (2) Might the resulting pure-structure computational motif manifest itself in familiar sequence-based computational motifs? RESULTS: The starting structure motif was a crystal structure of DNA bound to the integration host factor protein (IHF) of E. coli. IHF is known to exhibit both direct and indirect recognition of its binding sites. (1) Threading DNA sequences onto the crystal structure showed statistically significant partial separation of 60 IHF binding sites from random and intragenic sequences and was positively correlated with binding affinity. (2) The crystal structure was shown to be equivalent to a linear Markov network, and so, to a joint probability distribution over sequences, computable in linear time. It was transformed algorithmically into several common pure-sequence representations, including (a) small sets of short exact strings, (b) weight matrices, (c) consensus regular patterns, (d) multiple sequence alignments, and (e) phylogenetic trees. In all cases the pure-sequence motifs retained statistically significant partial separation of the IHF binding sites from random and intragenic sequences. Most exhibited positive correlation with binding affinity. The multiple alignment showed some conserved columns, and the phylogenetic tree partially mixed low-energy sequences with IHF binding sites but separated high-energy sequences. The conclusion is that deformation energy explains part of indirect recognition, which explains part of IHF sequence-specific binding.     

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding.

A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.     

Structural and functional organization of a porcine gene coding for nuclear factor I

This paper describes the structure of a 70-kb porcine gene for nuclear factor I, including its promoter region, comprising a total of 11 exons. Different mRNAs that we have isolated as cDNAs from both porcine liver and human HeLa cells presumably are generated from this gene by differential splicing events. One cDNA species from porcine liver that lacks exon 9 carries coding information for a protein of 439 amino acids. The in vitro translated protein displays all the properties of an NFI-like protein with high affinity toward the sequence element TGG(N)6GCCAA, as shown by gel shift analysis, and no or little affinity toward CCAAT box containing sequences. Cotranslation experiments with full-length and truncated variants of the protein demonstrate that it binds as a dimer to its cognate DNA recognition sequence. Its DNA-binding domain which is retained in all cDNA clones was mapped by deletion analysis to the 250 N-terminal amino acids of the protein. No structural homologies are observed between this protein and other known DNA-binding proteins; instead, the protein contains a novel alpha-helical sequence motif consisting of several lysine residues spaced at intervals of seven amino acids which we have termed the "lysine helix". The C-terminal portion of the protein derived from full-length cDNAs encodes a short amino acid sequence which is identical with the heptapeptide repeat CT7 observed in the C-terminal domain of the largest subunits of yeast and mouse RNA polymerase II. This region is removed by differential splicing in some of the NFI/CTF cDNAs and thus may be of functional significance.     

A single amino acid can determine the DNA binding specificity of homeodomain proteins

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.     

A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.     

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with -10 region single-stranded DNA.

Thesigmasubunit of RNA polymerase determines promoter recognition and catalyzes DNA strand separation. The -35 promoter region is recognized by a helix-turn-helix motif in region 4, while the -10 region is specified, at least in part, by an amphipathic helix in region 2. We have proposed that conserved aromatic residues insigmaregion 2.3 interact with the non-template strand of the -10 element to drive open complex formation. We now report that Bacillus subtilis sigmaA holoenzyme, but neither core nor sigmaA alone, binds with high selectivity to single-stranded (ss) DNA containing the non-template -10 consensus sequence. UV irradiation of holoenzyme-ssDNA complexes efficiently crosslinks sigmaA to DNA and protease mapping supports a primary contact site in or near region 2. Several mutations in sigmaA region 2.3, shown previously to impair promoter melting, affect ssDNA binding: Y184A decreases binding selectivity, while Y189A and W193A decrease the efficiency of photocrosslinking. These results support a model in which these aromatic amino acids are juxtaposed to ssDNA, consistent with their demonstrated role in stabilizing the open complex.     

Lactose repressor hinge domain independently binds DNA

The short 8–10 amino acid “hinge” sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer‐binding domains. Structural studies of full‐length or truncated LacI‐operator DNA complexes demonstrate insertion of the dimeric helical “hinge” structure at the center of the operator sequence. This association bends the DNA ～40° and aligns flanking semi‐symmetric DNA sites for optimal contact by the N‐terminal helix‐turn‐helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1–50 to remove the HtH DNA binding domain or residues 1–58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix‐turn‐helix domain with its highly positive charge. LacI missing residues 1–50 binds to DNA with ～4‐fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1–58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.     

Extrinsic interactions dominate helical propensity in coupled binding and folding of the lactose repressor protein hinge helix

A significant number of eukaryotic regulatory proteins are predicted to have disordered regions. Many of these proteins bind DNA, which may serve as a template for protein folding. Similar behavior is seen in the prokaryotic LacI/GalR family of proteins that couple hinge-helix folding with DNA binding. These hinge regions form short alpha-helices when bound to DNA but appear to be disordered in other states. An intriguing question is whether and to what degree intrinsic helix propensity contributes to the function of these proteins. In addition to its interaction with operator DNA, the LacI hinge helix interacts with the hinge helix of the homodimer partner as well as to the surface of the inducer-binding domain. To explore the hierarchy of these interactions, we made a series of substitutions in the LacI hinge helix at position 52, the only site in the helix that does not interact with DNA and/or the inducer-binding domain. The substitutions at V52 have significant effects on operator binding affinity and specificity, and several substitutions also impair functional communication with the inducer-binding domain. Results suggest that helical propensity of amino acids in the hinge region alone does not dominate function; helix-helix packing interactions appear to also contribute. Further, the data demonstrate that variation in operator sequence can overcome side chain effects on hinge-helix folding and/or hinge-hinge interactions. Thus, this system provides a direct example whereby an extrinsic interaction (DNA binding) guides internal events that influence folding and functionality.