Abstract
The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies. Mot1 is an essential regulator of RNA polymerase II-dependent transcription in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extracts. The preparation efficiently dissociated TBP.TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globular monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was stimulated about 10-fold by high ionic strength or alkaline pH, or by deletion of the N-terminal TBP-binding segment, suggesting that the N-terminal domain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondingly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimulated >/=10-fold by yeast TBP, suggesting that interaction with TBP relieves a conformational constraint in Mot1. Double- or single-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or Mot1C, with or without TBP. Mot1 did not exhibit detectable helicase activity in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequence. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwinding.
Highlights
¶ To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, Rm. 401, Barker Hall, University of California, Berkeley, CA 94720-3202
MOT1-(His)6 was expressed under the transcriptional control of the strong inducible GAL1 promoter in S. cerevisiae strain SC295 [40], which carries the reg1-501 mutation that prevents glucose repression of GAL-dependent gene expression [53]
Effect of TATA box-binding protein (TBP) and DNA on Mot1 Activity—Because Mot1 in extracts is found in soluble complexes that contain TBP and other polypeptides (56 –58), and because we have shown that TBP can interact with Mot1 in the absence of any other yeast proteins [45], the effect of TBP on the ATPase activity of purified Mot1 was tested
Summary
Materials—All reagent chemicals were purchased from Sigma or other appropriate commercial suppliers, as specified. 20 Ci of [␣-32P]dGTP (3 Ci/mol; NEN Life Science Products) and 10 units of the Klenow fragment of E. coli DNA polymerase I (New England BioLabs) were added in a buffer (60 l total volume) containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, and 6.67 mM DTT. The radiolabeled DNA probe was separated from unincorporated radioactivity by gel filtration over a bed (l ml) of Sephadex G-25 (Amersham Pharmacia Biotech) equilibrated in TE Buffer containing 100 mM NaCl. The amount of labeled probe was quantitated by both scintillation counting and a dot-blot assay using ethidium bromide staining and a standard curve prepared with samples of DNA of known concentration [51]. The band corresponding to the radiolabeled, full-length, and cross-linked probe was eluted from the gel in TE and used for gel retardation experiments, as indicated
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