Testing for DNA tracking by MOT1, a SNF2/SWI2 protein family member

Abstract

Proteins in the SNF2/SWI2 family use ATP hydrolysis to catalyze rearrangements in diverse protein-DNA complexes. How ATP hydrolysis is coupled to these rearrangements is unknown, however. One attractive model is that these ATPases are ATP-dependent DNA-tracking enzymes. This idea was tested for the SNF2/SWI2 protein family member MOT1. MOT1 is an essential Saccharomyces cerevisiae transcription factor that uses ATP to dissociate TATA binding protein (TBP) from DNA. By using a series of DNA templates with one or two TATA boxes in combination with binding sites for heterologous DNA binding "roadblock" proteins, the ability of MOT1 to track along DNA was assayed. The results demonstrate that, following ATP-dependent TBP-DNA dissociation, MOT1 dissociates rapidly from the DNA by a mechanism that does not require a DNA end. Template commitment footprinting experiments support the conclusion that ATP-dependent DNA tracking by MOT1 does not occur. These results support a model in which MOT1 drives TBP-DNA dissociation by a mechanism that involves a transient, ATP-dependent interaction with TBP-DNA which does not involve ATP-dependent DNA tracking.

FIG. 1

MOT1 and TBP dissociate rapidly from DNA in an ATP-dependent reaction that does not require an unobstructed DNA end. (A) Experimental scheme for the reactions analyzed in panels B and C. (B) Analysis of MOT1-catalyzed TBP-DNA dissociation on a “blocked” template by gel mobility shift. The template used is diagrammed above the autoradiogram. The total length of the probe was 154 bp, with 18 bp extending upstream of the EcoRI site and 36 bp downstream of the lac operator. The bands corresponding to various protein-DNA complexes are indicated by arrows. Note that addition of ATP to DNA preincubated with TBP, MOT1, lac repressor, and EcoRI-Gln 111 (lane 11) leads to rapid formation of a protein-DNA complex which comigrates with the lac repressor–EcoRI-Gln 111–DNA complex (lane 9). (C) Analysis of the fate of MOT1 and TBP on the same template as in panel B by DNase I footprinting. The footprints of proteins bound to EcoRI, TATA, and lac operator sites are indicated by rectangles. The bracket on the right denotes the DNA upstream of the TATA box protected from DNase I digestion by MOT1 when added to a reaction mixture containing TBP in the absence of ATP (lanes 3, 7, and 11). Note that addition of ATP for 1 min caused loss of the TBP and MOT1 footprints (lane 4), and this result is unaffected by EcoRI-Gln 111 bound upstream of the TATA box (lane 8) as well as by the binding of both EcoRI-Gln 111 and lac repressor to sites on either side of the TATA box (lane 12). (D) DNase I footprinting analysis of MOT1 activity on a DNA template containing a lac operator 36 bp downstream of a TATA box. The experiment was performed as described for panel C except that unlabeled lac operator DNA (“cold DNA”) was added as indicated to verify that lac repressor does not dissociate from the lac operator on the footprinting probe over the time course of the MOT1-catalyzed reaction. (E) DNase I footprinting analysis was performed as described for panel C by using a radiolabeled probe containing an EcoRI site 42 bp downstream from a TATA box. To verify that EcoRI-Gln 111 remains stably bound to the EcoRI site over the course of the MOT1-catalyzed reaction, a molar excess of unlabeled EcoRI-containing DNA (“cold DNA”) was added as indicated. (F) DNase footprinting experiments were performed as described for panels D and E by using the same footprinting probe as for panel C; unlabeled EcoRI-containing DNA was added as indicated to verify that EcoRI-Gln 111 is not dissociated from its site upstream of the TATA box over the course of the MOT1-catalyzed reaction.

FIG. 2

Strategy for testing of models of MOT1 action by using a DNA template with tandem TATA boxes. (A) MOT1 interacts with TBP-DNA via interactions with both TBP and DNA upstream of the TBP-DNA complex. Under template commitment conditions, dissociation of the TBP molecule directly interacting with MOT1 but not the adjacent TBP molecule might occur by a mechanism involving a transient, ATP-driven power stroke. Alternatively, in the presence of ATP, interaction of MOT1 with TBP-DNA might be followed by engagement with the DNA template and dissociation of both TBP molecules by an ATP-dependent DNA-tracking mechanism. (B) One requirement of the tandem TATA experiment is that MOT1 is specifically targeted to only one of the two TBP-DNA complexes on the tandem TATA template. (C) One prediction is that MOT1 can disrupt TBP-DNA on this template only via interactions with DNA upstream of the 5′ TBP-DNA complex. Hence, upstream truncation of the DNA should permit TBP binding to both TATA boxes, but these complexes should be refractory to MOT1 action.

FIG. 3

Gel mobility shift analysis of protein-DNA complexes formed on tandem TATA templates. The sequences of the two DNA probes used are shown at the top; the TBP binding sites are boxed. The upstream site, TGTAAAAG, is recognized by the altered-specificity TBP, TBPm3, but not by wild-type TBP. TBPm3 and wild-type TBP bind to the downstream TATA box. The free DNA as well as protein-DNA complexes formed on these probes are indicated by arrows. In this depiction, the open circles represent TBP or TBPm3, the black oval is MOT1, the DNA probe is represented by the black horizontal line, and the TATA boxes are shown as rectangles. The amounts of purified MOT1 added to the reaction mixtures are in microliters; this preparation of MOT1 had an activity of about 1 unit/μl (1a).

FIG. 4

Template-committed MOT1 disrupts both TBPs bound to the tandem TATA template. (A) Experimental scheme. The radiolabeled DNA, shown on top (asterisk), was incubated with TBP (open saddle shapes) in one reaction, and a 10-fold molar excess of identical, unlabeled DNAs was incubated with TBP in a parallel reaction. Since the unlabeled reaction mixture contains a molar excess of DNA over TBP, this reaction mixture presumably contains unbound, singly bound, and doubly bound DNA molecules which, for simplicity, are not all shown. After 20 min, MOT1 (black oval) was added to the reaction mixture containing radiolabeled DNA, and then the two reaction mixtures were combined with ATP for 1 min. DNase was added for 1 min, and then the reactions were terminated and the reaction products were resolved on 8% sequencing gels (see Materials and Methods). (B) The indicated proteins were added as outlined for panel A with the exception that the competitor TBP-DNA was added at different points in the scheme, as indicated. No competitor was added to the reaction mixtures in lanes 1 to 4. (C) The template commitment footprinting experiment was performed as described for panel B except that the reactions were performed in duplicate and footprinting was performed with two different amounts of DNase I. Competitor TBP-DNA was not added to the reaction mixtures in lanes 1 and 2.

FIG. 5

Strategy for testing for DNA tracking by MOT1 with lac repressor as a reversible steric block. (A) DNA containing two TATA boxes (open rectangles) separated by 80 to 100 bp is incubated with TBP (open saddle shapes) and lac repressor (gray circle), which binds to a lac operator (gray rectangle) precisely positioned upstream of one TATA box to interfere with MOT1 (black circle) binding to its proximally positioned TBP. The other TBP-DNA complex remains available for MOT1 interaction. One configuration of these sites is shown, but an analogous DNA containing a lac operator positioned just upstream of the 5′ TATA box was also tested (see text). Template commitment footprinting experiments were then performed in a fashion analogous to those in Fig. 4, with the exception that IPTG and unlabeled lac operator DNA were added along with unlabeled competitor TBP-DNA and ATP to induce the rapid dissociation of lac repressor from the operator on the footprinting probe. Following DNase treatment, the reactions were analyzed as described for Fig. 4 to determine if MOT1 bound to one TBP-DNA complex can influence the rate of dissociation of a second TBP-DNA complex on the same template without prior dissociation. (B) lac repressor bound to a lac operator positioned 12 bp (lanes 1 to 10) or 9 bp (lanes 11 to 18) upstream of a TATA box can block MOT1 action. TBP and lac repressor were incubated with radiolabeled DNA as indicated, and then MOT1 in the presence or absence of ATP and/or IPTG plus unlabeled lac operator was added for 1 min followed by addition of DNase I and analysis of the reaction products, as described for Fig. 4. The regions of DNA protected by TBP and lac repressor are indicated (rectangles), and the upstream extension of the TBP footprint induced by MOT1 in the absence of ATP is indicated by a bracket.

FIG. 6

Testing for ATP-dependent DNA tracking by MOT1 in the 5′-to-3′ direction by DNase footprinting. (A) The footprinted regions centered over the indicated sites are shown as open rectangles, and the distances between the sites are shown in base pairs. The indicated proteins were added to the probe as schematized in Fig. 5A, with the exception that the unlabeled competitor TBP-DNA was added in the order indicated at the top of the figure. For the reactions in lanes 1, 2, 8, 9, and 12, the competitor reaction mixture was added after preincubation with the indicated proteins and immediately prior to the addition of DNase. The unmarked arrow indicates a position upstream of the 5′ TATA box which is protected by MOT1 in the absence of ATP (lanes 6 and 11). (B) Comparison of the effects of MOT1 on TBP-DNA complexes subject to the reversible lac repressor steric block on templates with (lanes 1 to 14) or without (lanes 15 to 18) the second 5′ TATA box. Experiments were performed as described for panel A; the unlabeled TBP-DNA competitor was added to the reaction mixtures in lanes 1, 2, 5, 8, 11, 14, 15, and 16 after preincubation with the indicated proteins and immediately prior to addition of DNase.

FIG. 7

Testing for ATP-dependent DNA tracking by MOT1 in the 3′-to-5′ direction by DNase footprinting. (A) The binding sites and footprinted regions on DNA are indicated as in Fig. 6. The brackets indicate the region on DNA protected by MOT1 in the MOT1-TBP-DNA ternary complex formed in the absence of ATP. The experiment was performed as described for Fig. 5A and 6. Unlabeled TBP-DNA competitor was added to the reaction mixtures in lanes 1, 2, 5, 8, 9, and 12 after preincubation with the indicated proteins and immediately prior to the addition of DNase. The results shown in lanes 13 to 24 are from the same experiment as for lanes 1 to 12, but the film was exposed longer in order to better visualize the partial DNase protection upstream of the 5′ TATA box (brackets) afforded by MOT1, which has translocated along DNA from its loading site at the downstream TATA box (see text). (B) Control DNase footprinting experiment for panel A which demonstrates that EcoRI-Gln 111 binding to the EcoRI site (lanes 5 to 7) does not interfere with MOT1 action at either of the TBP-DNA complexes formed at the two TATA boxes in the absence of lac repressor. The template used in this experiment was the same as that used for panel A.