The 5.5 Protein of Phage T7 Inhibits H-NS through Interactions with the Central Oligomerization Domain

The 5.5 protein (T7p32) of coliphage T7 (5.5_T7) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5_T7 protein is insoluble when expressed in Escherichia coli, but we find that 5.5_T7 can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5_T7 binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5_T7 can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5_T7 protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5_T7 protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression.     

Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.     

Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.

To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.     

Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity

We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity. Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown. However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type. The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation. It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719). The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity. Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate.     

Analysis of oestrogen receptor dimerisation using chimeric proteins.

Sequences essential for dimerisation have been identified in the hormone binding domain of the mouse oestrogen receptor by insertional and point mutagenesis and sequence comparisons reveal that equivalent residues may be conserved in other members of the nuclear hormone receptor superfamily. To assess functional compatibility of this region between members of the receptor superfamily, peptide sequences corresponding to the equivalent regions of the human androgen receptor and retinoic acid receptor have been substituted for the dimerisation domain of the mouse oestrogen receptor. The resulting chimeric proteins were analysed for high affinity DNA binding using a gel retardation assay and shown to bind with reduced affinity compared to the wild type oestrogen receptor. The reduction in DNA binding observed may result from the intramolecular incompatibility of functional elements within the hormone binding domain of nuclear hormone receptors.     

Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity.

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)] appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is involved in nonspecific DNA binding, we found that up to 96% of this latter activity was specifically inhibited in wild-type T antigen by several monoclonal antibodies which collectively bind to the region between residues 269 and 522. In order to investigate the relationship between the origin-binding domain and the second region, we performed origin-specific DNA binding assays with increasing amounts of calf thymus DNA as competitor. The results suggest that this second region is not an independent nonspecific DNA binding domain. Rather, it most likely cooperates with the origin-binding domain to give rise to wild-type levels of nonspecific DNA binding. Our results further suggest that most of the nonspecific binding to double-stranded DNA is involved in a function other than direct recognition and binding to the pentanucleotides at the replication origin on simian virus 40 DNA.     

Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.     

The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria

The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae . Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans -activator protein in Rhodobacter capsulatus , has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli . Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides , XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae . These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.     

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.     

An Escherichia coli protein that preferentially binds to sharply curved DNA

We attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay. Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature. We purified such a protein from E. coli. Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences. The purified protein was identified as the E. coli nucleoid protein, H-NS.     

Binding of low mobility group proteins with DNA examined in homologous and heterologous systems by Gel retardation assay.

The interaction of low mobility group proteins (LMG), isolated from chromatin of pancreatic carcinoma cells (CAPAN-2), with fragments of 5′-flanking region of the antigen 17-1A gene was studied by gel retardation assay. The LMG proteins, which formed complexes with DNA were extracted from the gels and identified by polyacrylamide gel electrophoresis under denaturing conditions. The proteins of Mw about 100, 60, 55 and 48 kDa, which formed specific complexes with fragments of 5′-flanking region of the antigen 17-1A gene, were identified.     

Single exchanges of amino acids in the basic region change the specificity of N-Myc.

We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity. The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins. Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments. For this we used palindromic target sequences with systematic base pair exchanges. Several mutants with altered DNA binding specificity were identified. Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)). The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are. Exchanges at position -15 broaden the binding specificity. These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence.