Caracterización de los lugares de práctica

From in vitroexperiments, it was reported that Mot1’s ATPase activity directly dissociates TBP from DNA [161, 171, 165]. However, in the experiments de- scribed in the previous sections, no dissociation of TBP from the TATA-box could be observed. The influence of Mot1/TFIIA is reflected in the change of the transitions rates for the highly dynamic TBP/DNA complex as reported in Section 5.2.2. To verify the dissociation activity of Mot1 on a single-molecule level, the binding of single TBP molecules to the TATA-box under the influence of Mot1 was observed. The binding of TBP to DNA was monitored by measur- ing the fluorescence intensities of the fluorescently labeled TBP molecules. As long as fluorescence could be observed, the TBP was assumed to be bound to the DNA. Disappearance of the fluorescence signal after ATP addition can be due to two factors: photobleaching (see Appendix A.4) or dissociation of TBP from the DNA. The rate for photobleaching of a donor molecule depends on the time that a donor molecule spends in the excited state and in turn on the FRET value. From the experiments in Section 5.2.1, we know that ATP addition in- duces a significant change in the FRET efficiency and in turn will alter the rate of donor bleaching. To avoid any influence of FRET on the rate of donor pho- tobleaching, TBP was labeled as acceptor in the experiments described in this section.

Experiments and Results

The preincubated ternary complex, TBP/Mot1/DNA, were immobilized at the surface and unbound complexes were washed away by flushing with buffer. This washing step was performed with working buffer and removed free Mot1 from the sample chamber. In contrast to ensemble measurements, where unbound TBP and Mot1 are omnipresent, in this experiment two different initial condi- tions can be established. The experiment can be performed without any residual protein in solution by removing them with a flushing step or by adding Mot1 to the buffer that is used to add ATP. This way a reproducible amount of unbound Mot1 is added to the reaction chamber. The different experimental conditions

are illustrated in Figure 5.19.

Prior to each dissociation experiment, the characteristic photobleaching time was determined4. ThisTime Before Disappearance (TBD) was used to account for photobleaching and to identify any additional contributions to the disappear- ance of the fluorescence.

The experiment was prepared in such a way that the recording was started before addition of ATP to the flow chamber and care was taken that ATP addition did not cause focus drift. The effect of ATP addition to a sample preparation in the flow chamber can only be recorded once per ATP addition. This limits the number of molecules which are available for the analysis. The number of molecules available for analysis was increased by doubling the field of view and recording the fluorescence from the red channel only. The first 10 frames were recorded during excitation of the Atto532 labeled DNA with532 nm light. The fluorescence recorded in the red channel during this first 10 frames was due to FRET between the donor labeled DNA and the acceptor labeled TBP. This first 10 frames were used to identify the complexes where TBP was bound to the TATA-box. Only molecules where fluorescence in the red channel due to FRET could be observed, where selected for analysis. After the first 10 frames and before ATP addition the excitation was switched to647 nm.

The decay in the number of TBP/Mot1/DNA complexes over time for both ad- dition schemes (ATP ± Mot1) are shown in Figure 5.20. The frames before ATP addition are removed and the decay normalized.

The previous experiment was repeated but without Mot1 preincubation. The binary TBP/DNA complexes were immobilized and ATP together with Mot1 added. No significant dissociation could be observed as shown in Figure 5.21. The results were fitted to a mono-exponential decay. The rates obtained from the experiments with ATP and Mot1 addition were corrected by subtracting the rate of photobleaching. The resulting rate was measured as a function of ATP concentration as shown in Figure 5.22.

4_{Significant variations of the photobleaching time between different flow chambers were observed, which makes}

5.2. Interaction of Mot1 with the TBP/DNA binary complex 125

+

+

A

B

Figure 5.19.: Illustration of the Mot1-catalyzed TBP-DNA dissociation. A: Reaction scheme

without free Mot1 in solution by removing any unbound proteins by flushing with buffer prior to ATP addition. B: Reaction scheme with Mot1 by flushing the reaction chamber with a Mot1 and ATP containing

0 5 10 15 20 25 0 0.2 0.4 0.6 0.8 1 time [s]

normalized number of molecules

0mM ATP 1mM ATP data3 data4 data5 data6 0 5 10 15 20 25 0 0.2 0.4 0.6 0.8 1 time [s]

normalized number of molecules

1mM ATP 0mM ATP 0mM ATP 0mM ATP

Figure 5.20.: Kinetics of Mot1-catalyzed TBP-DNA dissociation measured by the decay of

number of fluorescent molecules. Left: addition of 1mM ATP to DNA/TBP/Mot1 ternary complexes. The red curve shows the decay of number of fluorescent molecules due to photobleaching (without ATP), the blue curve shows the decay in the number of fluores- cent molecules after ATP addition. Right: same experiment as before but with the addition of1 mMATP together with3.4 nmMot1.

Figure 5.21.: Kinetics of Mot1-catalyzed TBP-DNA dissociation measured by the decay of number of fluorescent molecules. Addition of 1 mM ATP together with 3.4 nm Mot1 to preincubated binary TBP/DNA complexes. In contrast to Figure 5.20 no significant dissociation is observed if TBP is not preincubated with Mot1.

2 4 6 8 10 x 10−4 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 ATP concentration [M] rate [sec − 1]

5.2. Interaction of Mot1 with the TBP/DNA binary complex 127

Discussion

The Mot1 ATPase activity does not directly dissociate TBP from DNA when no free Mot1 is available in solution. TBP is liberated only in the presence of free Mot1 in solution. The results of these experiments are in agreement with

in vitro experiments where TBP displacement by Mot1 ATPase activity was

demonstrated [161]. The important difference between previous ensemble mea- surements and the single-molecule data is the presence or absence of free Mot1 in solution. The rate of the TBP dissociation is ATP and Mot1 concentration dependent. The lack of TBP dissociation could be a result of insufficient Mot1 binding during the preincubation. This can be ruled out since Mot1 readily binds the binary TBP/DNA complex as shown in Figure 5.3, where the majority of molecules undergo a conformational change during Mot1 preincubation. Whether or not the complexes after ATP addition (without free Mot1 in solu- tion) still have Mot1 bound cannot be judged from these experiments alone. Two lines of evidence support a model in which Mot1 is still bound to the TBP/DNA complex after ATP addition: In the experiments with double-labeled DNA (see Section 5.2.2), an altered DNA dynamic after ATP addition was observed, sug- gesting that Mot1 is still present after ATP addition and, in the three-color ex- periments (see Figure 5.4), Mot1 could still be detected after ATP addition.

The differences in Mot1 ATPase activity for experiments with and without free Mot1 in solution can be explained by two models: Mot1 dissociates TBP from the TBP/binary complex during ATPase activity with a low efficiency. In this framework, with only ATP in the solution, Mot1 has only one round of ATPase hydrolysis to liberate TBP. Rebinding or binding of another Mot1 molecule is very unlikely due to the low local concentration of Mot1 molecules available at the prism surface. If Mot1 is supplied in the solution together with ATP, re- peated rebinding is possible and, despite the low dissociation efficiency, TBP liberation becomes possible. An other possibility is that the ATPase activity of Mot1 does not dissociate TBP from DNA but rather modulates and weakens the binding. In this model, Mot1 does not necessary need to be displaced from TBP to explain the lack of TBP dissociation activity. If additional Mot1 is avail-

able in solution, it dissociates TBP by interactions with the "primed" TBP/DNA complex. In this model, Mot1 has two distinct functions, modulation of the TBP/DNA conformation and dissociation of the primed TBP/DNA complex. It is important to note that, in this model, more than one Mot1 molecule is required for TBP liberation.