Purification and Enzymic Properties of Mot1 ATPase, a Regulator of Basal Transcription in the Yeast Saccharomyces cerevisiae

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 TBP⅐TATA 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 Transcription by all three eukaryotic RNA polymerases requires TBP.1 For initiation by yeast Pol II, TBP binding to a TATA sequence present in most promoters nucleates stepwise assembly of a large complex containing TBP-associated factors (1), termed TFIID, as well as multiple general transcription factors and Pol II (reviewed in Refs. 2-5). In an alternative, but not mutually exclusive, model of initiation, TFIID binding creates a platform for recruitment of a massive holoenzyme, consisting of Pol II, the SRB proteins, and other cofactors (like Gal11), as well as the general transcription factors, TFIIB, TFIIF, and TFIIH (reviewed in Refs. 6 -8). In both models, promoter binding by TBP is the rate-limiting step in initiation complex formation and thus provides a physiologically relevant target for transcriptional control by both positive (9, 10) and negative (11-14) regulatory factors.Factors that negatively regulate eukaryotic transcription utilize a wide variety of mechanisms, including quenching of activators, promoting assembly of chromatin, recruiting chromatin-binding proteins or histone-modifying enzymes, and interfering with the general transcription machinery itself. In addition, several proteins are thought to block Pol II transcription via interaction with TBP. Such regulators include Evenskipped (eve gene product), a Drosophila homeodomain protein (15, 16), and a general repressor, NC2 (or Dr1-DRAP1) (17-19). NC2 binds to TBP⅐DNA complexes and prevents the association of TFIIA and TFIIB, possibly by altering conformation of the .Another kind of negative regulator of TBP action was first identified both genetically and biochemically in the yeast Saccharomyces cerevisiae. A TBP-targeted repressing activity, termed ADI for "ATP-dependent inhibitor" of TATA binding, was detected in yeast nuclear extracts (25). ADI removed TBP from the TATA element of the adenovirus major late promoter (AdMLP) in an ATP-dependent manner. TFIIA, and to some extent TFIIB, inhibited ADI activity by competing for binding to TBP or by stabilizing the TBP⅐DNA complex, or both (25). It was subsequently found that ADI corresponded to the product of the MOT1 gene (26). The MOT1 locus had been previously identified by temperature-sensitive mutations that increased basal expression of many Pol II-specific genes (27, 28).The MOT1 gene was isolated by complementat...

Two-step Mechanism for Modifier of Transcription 1 (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA

“…1B) (42). Moreover, although the ATPase activities of other Snf2/Swi2 family members are stimulated by DNA (37), the ATPase activity of Mot1 is stimulated by TBP and not by DNA alone (44,45). Nevertheless, a sequence alignment with other enzyme family members reveals that many residues implicated in DNA binding are conserved in Mot1 (40), and mutational analysis indicates that at least some are functionally important for the catalytic action of Mot1 (43).…”

“…Mot1-TBP complexes have relaxed sequence specificity for DNA (36), but the cause of this altered behavior is unclear. In contrast to other Snf2/Swi2 family members (37)(38)(39)(40)(41), full-length Mot1 has no detectable DNA binding affinity (16,36,42,43), and the ATPase activity of Mot1 is stimulated by TBP in vitro (44,45). However, biochemical evidence indicates that the ATPase domain is in close proximity to DNA when Mot1 is assembled with TBP-DNA (35,43), suggesting direct Mot1-DNA interaction.…”

“…Note that the data were best fit with a Hill coefficient of 2. Biochemical and structural data indicate that Mot1 has a single ATP binding site and functions as a monomer (35,45), so the significance of the Hill coefficient is unclear. of the SsoRad54 ATPase reveals that the Snf2/Swi2 ATPase domain is composed of two RecA-like motifs, with DNA binding along the interface between these two motifs (40, 66).…”

“…Evidence that the ATPase activity of full-length Mot1 is stimulated by TBP (but not DNA alone) (44,45) Table 2. Note that the data were best fit with a Hill coefficient of 2.…”

“…4, A-E). Given the millimolar concentrations of ATP in the yeast cell (68) and the K m of Mot1 for ATP (ϳM) (36,43,45), one could suppose that Mot1 is typically bound to ATP in vivo. Therefore, in the cell, Mot1 might contact TBP-DNA complexes as Mot1-ATP (step 3).…”