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Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase. Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (∼120 kDa) of the protein complex. We assigned all of the 13C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques. The result of 13C-carbonyl NMR experiment on [13C′-Met]-CRP in the presence of both cyclic AMP and RNA polymerase α subunit showed that the two proteins interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP. The results also showed the effectiveness of the selective labeling and 13C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules. [ABSTRACT FROM AUTHOR]

The amino-terminal domain of the a subunit (αNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the α, β and β′ subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of αNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, αNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances. Analysis of the pattern of NOEs observed between the backbone amide and a-protons demonstrated that αNTD has three α-helices and two β-sheets. Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two β-sheets has two additional β-strands. The irregular NOE patterns observed for the three positions in the β-sheets suggest the presence of β-bulge structures. The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly. [ABSTRACT FROM AUTHOR]

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-ter-minal subdomain with a positively charged cleft in a novel α/β structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (D/D) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-tenninal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA. [ABSTRACT FROM PUBLISHER]

The region containing the RNA polymerase a subunit (RNAPα) gene (rpoA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Southern hybridization. The gene order in this region is rpl36-rpsl3-rpsll-rps4-rpoA-rpll7, which is identical to that in some other eubacteria. The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 35,013, the amino acid sequence showing 42% identity to that of Escherichia coir (Ec). From the results of comparison of the amino acid sequence and the predicted secondary structure of the C-terminal domain of Tt RNAPα (Tt αCTD) with those of Ec, the overall folding is expected to be similar. However, amino acid residues Asn268 and Cys269 in Ec αCTD, which are essential for its interaction with DNA or regulatory proteins, were replaced by His and Ser, respectively, in Tt αCTD. By means of a T7–based expression system in Ec cells, Tt RNAPα was overexpressed and purified. The high thermostability of Tt RNAPα was demonstrated by the CD spectra. [ABSTRACT FROM PUBLISHER]

Eight synthetic Holliday junction (HJ) oligonucleotides containing an immobile or a mobile junction were characterized by gel electrophoresis, ultraviolet absorption and circular dichroism (CD) spectroscopy. Four 24-mer deoxyribonucleotides formed stable immobile and mobile HJs in 0.1 M NaCl at 5 μM strand concentration at room temperature. However, the immobile HJ constructed from four 18-mers was less stable, and four 12-mers did not form the HJ structure under the conditions used. A comparison of the melting profiles of the HJs with those of the duplexes corresponding to the arms of four-way junctions indicated that the thermal stability of the HJ was similar to that of the individual arm and the cooperativity of the melting behavior of the HJ was relatively higher than that of the individual arm duplex. The Tms of the mobile HJs containing 4,6,8, and 10 base-pair homologous cores at junctions were essentially identical with that of the immobile HJ of the same size. There is a tendency that the HJ containing a larger homologous core region becomes more resistant to thermal denaturation. The addition of divalent metal cations, Mg2+ and Ca2+, to the solutions of the HJs raised their melting temperatures. The difference found for the CD spectra of the HJs which differ only in the arrangement of the HJ depended primarily upon the DNA sequence flanking the junction. The RuvC protein binds to the immobile and mobile HJs, regardless of the presence and the size of the homologous core at the junction. [ABSTRACT FROM AUTHOR]