Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda:Rhabditida)

We have calculated the curvature of 504 eukaryotic promoters predicted by the bent A-tract model of Bolshoy et al. (Proc. Natl. Acad. Sci. USA, 88(6), pp. 2312-16) and the bent non-A-tract models of Calladine et al. (J. Mol. Biol., 201, pp. 127-37) and Satchwell et al. (J. Mol. Biol., 191, pp. 659-75) and found in each case a correlation between TBP binding sites and DNA curvature. Characterizing the TBP binding sites revealed that in addition to the classical TATA box (TATAAA) five more elements occur significantly often in the promoters, nearly all of them being one point mutations of the classical TATA box element. Separate curvature calculations for promoters with canonical and non-canonical TATA boxes have shown that in both cases the strong curvature of the helix axes in the domain of the binding sites is maintained (classical TBP binding sites: + 64-135%, non-classical TBP binding sites: + 27-49%). These results support the proposition that beside DNA flexibility and DNA-protein interactions intrinsic curvature of DNA is one further important criterion for the recognition of different DNA elements by TBP.     

Architecture of protein and DNA contacts within the TFIIIB-DNA complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding

The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables.     

Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription

We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae. Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX. Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation. The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2. Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box. The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells. The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.     

Binding and catalytic properties of Xenopus (6-4) photolyase

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.