Optical Tweezers: Single Molecule Manipulation in Biochemistry

Top

00:00:00.25 Good day. My name is Carlos Bustamante, and today we are going to talk
00:00:04.25 about the use of single molecule methods
00:00:08.04 specifically single molecule manipulation methods in biochemistry.
00:00:11.25 In the first half we are going to talk about why do we want to use single molecules
00:00:17.24 and some of the rationale of how do we, why do we actually study these reactions at the single molecule level,
00:00:25.19 what instrumentation is used to do that kind of experiments,
00:00:31.05 and in the second half I am going to present you a particular application of this method
00:00:35.25 of single molecule manipulation to study a molecular motor.
00:00:40.00 I want to start by reminding you that most of chemistry and biochemistry has been done by using the ensemble approach.
00:00:53.25 In the ensemble approach, essentially you can follow a chemical reaction
00:00:57.28 between component A and a component B to produce a component C
00:01:02.02 as indicated here in this reaction.
00:01:04.18 And the idea is that you mix a very large number of molecules of A with a very large number of molecules of B,
00:01:10.26 to produce also a very large number of molecules of C.
00:01:14.10 In fact, I indicate here that typically we work with the order of Avogadro's number of molecules, 10 to the 24 molecules.
00:01:21.27 And I want to in the first movie here you will see that in fact that is essentially what we do when we do a titration,
00:01:29.21 for example in the lab in Chemistry 1.
00:01:32.04 You have the compound with a particular color at the beginning. You add the second compound,
00:01:37.03 and as a result of the reaction the mixture actually changes color and again you add yet another reactant,
00:01:47.20 and again the mixture changes color once again.
00:01:50.17 indicating that the reaction is proceeding.
00:01:52.11 Essentially you are following the reaction by the change of this global ensemble property of the solution.
00:02:00.07 Now this is what we call the ensemble approach, and also it is called bulk methods of studying chemical reactions.
00:02:12.28 And in this particular talk we are going to refer to them as well as studies made in multiplo.
00:02:19.29 to indicate that we use many molecules to actually follow the reaction.
00:02:25.02 Now there are many advantages to this way of studying reactions in bulk.
00:02:30.14 And it is first that it is a very robust method because the signals are proportional to the number of molecules,
00:02:37.03 and we've seen that there are actually lots of molecules reacting A+B to produce C.
00:02:43.18 On the other hand we must remember that what we are doing is we are following in this case
00:02:50.02 what happens to the population on average.
00:02:53.02 See, in this case we are following that by the change in one property.
00:02:59.29 In our particular example, in the movie that you just saw,
00:03:02.27 that property is the color of the solution.
00:03:05.08 And we see how that color of the solution changes over time
00:03:09.10 and that change is essentially change that we are following over the whole population of molecules.
00:03:15.26 Now in reality of course, if we had a microscope, that would allow us to enter the solution and see the individual molecules,
00:03:26.15 we would find a completely different picture.
00:03:28.25 Every molecule would be doing something very different.
00:03:32.22 For example, they would be reacting some very fast. Some of them would be reacting at some intermediate rate,
00:03:39.13 and some would be reacting very, very slow.
00:03:42.20 And so, still what we follow of course is the average of these molecular behaviors.
00:03:48.29 And yet we can learn quite a bit about the reaction.
00:03:54.04 In fact, there are, however, limitations to this approach.
00:04:02.25 Right? Because despite the power and the robustness we actually have to compromise quite a bit
00:04:10.29 in order to study a chemical reaction in bulk, as an ensemble, in multiplo.
00:04:16.18 And is that in reality we are not following the molecular trajectories, the trajectories of the individual molecules
00:04:24.15 as they carry out the reaction.
00:04:26.10 Instead what we are actually doing is we are following the dynamics of the mean of the population.
00:04:31.13 Something that we actually call in chemistry kinetics.
00:04:35.11 Okay. Now in reality what we would like to do is to be able to describe
00:04:43.10 and to see how the different molecules in the solution are actually carrying out the reaction.
00:04:49.10 But that requires that instead of studying the system, the molecules, the reaction,
00:04:55.18 as an ensemble of molecules, that we actually follow the reaction one molecule at a time.
00:05:03.00 So you may ask why do we want to follow reactions one molecule at a time.
00:05:08.11 Are there any advantages in doing that relative to the ensemble or bulk approach.
00:05:16.21 Well, I would like to point out to you that one place in which following reactions
00:05:26.11 with a very few number of molecules or following reactions at the single molecule level
00:05:32.08 is important is the interior of the cell.
00:05:35.24 In fact, many cellular processes we know now such as, for example, chromosome segregation
00:05:41.22 chromosome replication, transcription of DNA, translation, recombination,
00:05:48.13 are often carried out by very few molecules in the cell.
00:05:55.14 So here is an interesting back of the envelope calculation.
00:05:58.24 What you see here is a cell of E. coli.
00:06:02.04 And you may ask, given the volume of an E. coli cell,
00:06:06.27 imagine that you have a single molecule of any kind
00:06:12.16 indicated here in this yellow square inside this E. coli cell.
00:06:18.00 And now you ask, what is the concentration of that single molecule of that particular type inside that E. coli cell?
00:06:27.13 Because the volume of the E. coli cell is so small, it turns out that one molecule in an E. coli cell
00:06:34.00 is at the concentration already of 1.6 nanomolar.
00:06:38.23 Right. And that is a very interesting result because this suggests that many reactions can occur
00:06:48.16 inside the cell with just having one single molecule of any kind inside the bacterium. Right?
00:06:57.03 In fact, imagine that that protein is a DNA binding protein.
00:07:03.21 We know that the affinity of most DNA binding proteins is below nanomolar.
00:07:08.21 Therefore the presence of one single molecule of that protein in the cell, given a concentration of 1.6 nanomolar,
00:07:18.15 assures that that protein will be 90 or 99 percent of the time bound to its target site on DNA.
00:07:26.04 In other words, reactions in the cell because of the tiny volume of the cell can occur
00:07:30.26 when the molecules are present at very, very small numbers.
00:07:33.28 Very, very small numbers.
00:07:36.05 So, why do we want to study single molecules or reactions at the levels of single molecules?
00:07:46.05 Well, there are many questions that we might want to answer if we are able to follow reactions at the single molecule level.
00:07:52.25 For example, first, how do reactions in the cell occur when you look at them one molecule at a time?
00:08:00.04 What are they telling us about how the mechanism of the reaction is... what the mechanism is?
00:08:09.00 We may want to also find out whether all of the molecules follow the same trajectory
00:08:14.11 every time we go from A to B or whether they follow different trajectories or different paths.
00:08:20.17 We also want to know perhaps how noisy
00:08:26.20 are the processes in which molecules are reacting one molecule at a time.
00:08:31.26 Remember, now we are looking at very few molecules
00:08:34.27 and you expect that there could be great fluctuations in the behavior of these molecules and in the signal.
00:08:40.20 And can we learn something about these fluctuations or is this just noise that contains no information?
00:08:48.00 So all of these questions are interesting questions that become accessible
00:08:52.01 if we develop methods that would allow us
00:08:54.28 to study chemical reactions one molecule at a time.
00:08:59.18 Of course we must be prepared to pay some price for studying single molecules.
00:09:05.14 These studies, which we can actually call in singulo
00:09:08.20 to indicate that we are now studying a reaction one molecule at a time,
00:09:13.26 have signals that are very different from the bulk signals that remember were very robust, very quiet, very nice.
00:09:23.02 In this case, your molecules will instead display very fast instantaneous dynamics,
00:09:27.13 which means that the behavior, the signal that you measure, the behavior of the molecules,
00:09:33.06 actually may appear random or sometimes stochastic.
00:09:37.09 In other words, very noisy.
00:09:39.07 You will see that fluctuations in this case dominate, in a sense, the magnitude of the signals.
00:09:44.24 And you may find that the molecules may be found sometimes to coexist in more than one state.
00:09:51.28 Sometimes they are in one state, sometimes they may actually go into a second state or a third state,
00:09:58.04 and they may be actually moving along this path, co-existing in these different states.
00:10:05.05 And even in some cases molecules may be in very unlikely states
00:10:09.14 but then all of a sudden they enter these unlikely states and you may observe them doing that.
00:10:16.04 This is what we call extreme states of the molecules.
00:10:20.02 But they're are part of the behavior of the molecule, and as such they are of interest, of course, for studying.
00:10:27.11 I want to illustrate some of the advantages of studying single molecules
00:10:36.04 by pointing out to you that as I mentioned before, molecules sometimes may not exist just in one single state,
00:10:43.24 but they may coexist in several states at equilibrium.
00:10:48.08 And so in fact the molecular property that you are interested in studying for example,
00:10:55.18 let's assume that there is the ability to absorb light or
00:11:00.04 the dimensions of the molecule, or for example the state of the molecule,
00:11:08.05 whether it is a monomer or forming a dimer or aggregated in some larger structure.
00:11:16.19 Whatever the molecular properties that you are studying may not be actually a single population of them,
00:11:24.10 but actually you may have a multiple population where you may have molecules spending some time in this type of property
00:11:34.00 with this particular value of the property or they may actually spend some time also in this other property.
00:11:41.02 And that there may be some kind of exchange between these two populations.
00:11:46.11 If you are using an ensemble method of course what you are going to get
00:11:50.26 is not information about the existence of this population or that population,
00:11:55.19 but typically what you will get is actually the average of the two populations.
00:11:59.15 That's the signal that you are going to measure, and the molecular property that you are going to actually be studying,
00:12:05.06 in this case, is going to be the value given by this green bar, as indicated here.
00:12:12.01 But it is also possible that the molecules actually exist in a single population
00:12:17.27 that is very broad as indicated here in this second panel.
00:12:21.23 In this case you may end up actually having the same average,
00:12:27.07 but now you have just a single population that has a rather broad distribution.
00:12:31.04 Or yet in the more extreme case you may have just a single population as indicated here,
00:12:39.09 but with the same value of average. So notice in fact that in these three panels,
00:12:46.13 the one on the left, the one in the center, the one on the right,
00:12:49.06 actually in bulk methods, in ensemble methods, will give you the same answer
00:12:55.09 even though the behavior of these molecules is very different because in one case
00:13:02.17 you have a multimodal population whereas in the other two cases you have actually a single population.
00:13:09.12 Okay, so what I would like to do is now motivate this study of single molecules
00:13:18.19 by telling you a little bit about how in my laboratory we started studying molecules one at a time.
00:13:26.07 And for that I am going to tell you a story about the elasticity of single DNA molecules.
00:13:31.15 Many years ago we were studying how molecules of DNA actually move inside a gel.
00:13:41.07 As you know electrophoresis is a very powerful technique that biochemists constantly use
00:13:46.21 to separate molecules of different size. Molecules of DNA of different size or molecules of proteins,
00:13:53.21 protein molecules of different size.
00:13:55.12 In this particular case, we wanted to know how is really that molecules of DNA separate by size.
00:14:02.14 In order to do that what we did was an experiment where we mixed molecules of DNA with ethidium bromide,
00:14:09.04 which is a dye that intercalates in the DNA and that renders the molecules fluorescent.
00:14:14.08 Now we mix these molecules of DNA with a gel, in this case it was an agarose gel.
00:14:21.06 And then we put them between a slide and a coverslip, allowed the gel to harden,
00:14:27.13 and then we sealed this coverslip with nail polish. It is always good to actually have some nail polish around in the laboratory.
00:14:37.00 And then we applied two electrodes on both sides of the slide,
00:14:40.22 and we look at these slide coverslip type of chamber under the fluorescence microscope,
00:14:50.14 in an optical fluorescence microscope.
00:14:52.20 And what you see is what we saw is what you see in this movie.
00:14:56.20 As you can see actually the molecules of DNA, in this case these are molecules of T4, bacteriophage T4,
00:15:03.12 are moving all towards the positive electrode. Right.
00:15:08.08 But notice that the molecules are actually extending as they go to the positive electrode.
00:15:14.04 Both arms in the direction of the positive electrode,
00:15:15.01 but one of the arms is usually larger just by chance than the other.
00:15:20.17 It has more charge, more phosphates, and therefore experiences more force,
00:15:24.14 and it will win the battle and will end up actually pulling the other arm around the corner as indicated here.
00:15:31.06 There it goes. Notice how it pulls around the corner.
00:15:34.15 And the molecule actually collapses again to start a new cycle.
00:15:39.01 This repeats over and over, but what I would like you to notice is how when the collapse occurs
00:15:46.09 how elastic the molecule appears when the collapse occurs.
00:15:51.07 In other words, when that particular arm turns the corner, notice it catches up very fast.
00:15:56.16 And that really surprised us quite a bit.
00:15:59.15 In fact we knew about the elasticity, but seeing the elasticity at play actually was a great motivation,
00:16:05.26 and so we thought why not acutally try to understand the elasticity of a single molecule of DNA.
00:16:12.28 And so we decided to actually do an experiment to study the elasticity of a single molecule of DNA,
00:16:20.22 and the idea is quite simple.
00:16:21.22 As you see here in the slide, the idea is to attach one end of the DNA to a wall
00:16:28.24 and then grab the other end of the DNA and simply ask
00:16:32.29 how much force do I have to apply in order to extend the molecule a given amount.
00:16:37.07 That is essentially how we tried to understand what the elasticity of the molecule is.
00:16:43.17 Of course in practice, the way you do this experiment is by attaching the DNA to two microscopic beads
00:16:49.16 in this case plastic beads. One of the beads is kept atop a micropipette by suction as indicated here.
00:16:58.06 And that one, the second bead, and the other bead, which is the one you are going to apply the force to
00:17:04.28 actually is attached to the other end of the DNA.
00:17:10.10 And so the idea is then, again, we pull on this second bead out,
00:17:17.07 and we determine how much force we have to apply in order to extend the molecule by a given amount.
00:17:22.24 Now how are we going to apply the force?
00:17:26.21 There are many ways in which we can apply the force.
00:17:28.24 We could for example use a magnetic bead for the second bead, for this second bead.
00:17:33.02 And then bring a magnet near the microscope and in that way apply a magnetic force that will stretch the DNA.
00:17:40.26 Alternatively we can actually use what is called an optical tweezers instrument.
00:17:46.18 Optical tweezers were invented in 1970 and they were as a result of an observation
00:17:58.13 made by Arthur Ashkin many years ago, where he found that because light carries momentum,
00:18:04.27 it has the ability to trap objects that have a different index of refraction than the surrounding medium.
00:18:11.09 And so you have a beam intensity as shown here that is typically Gaussian,
00:18:15.15 for a laser, okay, so the light is more intense at the center than it is at the periphery of the beam.
00:18:23.19 Then you approach a bead close to the beam, what will happen is that that bead will experience a net force
00:18:32.00 as indicated here that will tend to suck up the bead towards the center of the beam.
00:18:37.19 Once that happens then every time you move the beam, the bead is going to be essentially...
00:18:43.24 the bead is going to follow the beam everywhere.
00:18:46.16 The bead is basically trapped in the beam of light.
00:18:51.26 And that is a great thing because you can actually now use light
00:18:56.16 to manipulate things far away. In other words, you can manipulate these microscopic beads as I showed you before.
00:19:03.18 And it is those microscopic beads happen to be attached to a molecule of DNA,
00:19:06.12 now you can use those beads and the beam of light to manipulate your molecules of DNA.
00:19:12.05 So, in fact in the next slide you can see how do we go about attaching the molecule of the DNA between the beads.
00:19:24.16 Because obviously that is a condition to do this experiment.
00:19:27.06 You see on top four panels which I have labeled A, B, C, and D.
00:19:32.29 So the idea is the following, what you do is you grab the molecule of DNA
00:19:36.26 and you modify the ends of the molecule of DNA, placing a biotin at both ends in this case.
00:19:43.26 Then you have beads that are covered with streptavidin which binds very, very tightly to biotin, as you know.
00:19:50.02 And then we place that bead atop a micropipette, as indicated in panel A.
00:19:55.15 And then we pass molecules of DNA by flow through the chamber.
00:19:58.26 Both ends of which are actually labeled with biotin, remember?
00:20:03.19 So as indicated here in the A panel, and the arrows indicate here that we are flowing the molecules of DNA.
00:20:11.00 As you can see here, right?
00:20:12.10 Now just by chance, we expect that one of the molecules of DNA will actually hit this bead,
00:20:18.08 and it will react the biotin on one end with the streptavidin on the surface.
00:20:24.13 Because we are applying flow, that molecule of DNA is going to be extended as shown here in panel A.
00:20:32.28 As you can see here. Okay, now that you have the molecule hopefully attached
00:20:37.16 now you pass more beads and you turn on the optical trap.
00:20:41.18 The optical trap or optical tweezers is indicated here by the tick marks.
00:20:45.22 The four tick marks.
00:20:47.10 One of the beads will pass by the trap and will get trapped in the optical tweezers trap, right in the center there.
00:20:54.07 And now what you do is, still in the presence of the flow,
00:20:58.06 because you want to maintain the molecule of DNA extended,
00:21:00.29 and knowing that that end of the DNA also has a biotin that can react with another streptavidin on the surface of that bead,
00:21:07.25 you then now approach by moving the pipette, the bead in the trap to the bead in the pipette,
00:21:16.22 encouraging the formation of a single bond as indicated here between the biotin at this end of the DNA and the bead.
00:21:26.16 And when that happens you should be able to actually pull the pipette
00:21:29.25 and in fact fish the bead out of the trap using the DNA as your fishing line.
00:21:36.08 Right. Okay, so that is the idea of this experiment.
00:21:39.19 And you will see in the movie here that we are already in panel B because we have a bead in the trap and a bead in the pipette.
00:21:49.00 Hopefully we have a molecule of DNA between the two,
00:21:53.15 but the binding between the second biotin and the streptavidin hasn't occurred yet.
00:21:58.08 But we are hoping that it will occur, and in fact there you can see that immediately that it happened.
00:22:04.04 So now you see that in fact we have now a molecule of DNA between these two beads.
00:22:09.11 And the process is so efficient in a sense that notice that actually we also hold another molecule of DNA
00:22:15.07 between that bead and another bead that was in the trap.
00:22:18.20 So in fact we have bead, DNA, bead, DNA, bead, right?
00:22:21.26 So it is a very efficient process and it only took a few seconds to actually put a molecule of DNA between two beads.
00:22:30.15 Okay, having the possibility of doing this allows us then to really pull the beads apart
00:22:37.14 and study how much force do I have to apply in order to extend the molecule a given amount.
00:22:43.05 And the result is shown here. This is what is called force extension curves of DNA.
00:22:48.05 And you can see that in the ordinate I am actually plotting here the force required to stretch the molecule,
00:22:57.06 and in the x-axis or the abscissa, I actually have here the extension, the relative extension of the molecule of DNA.
00:23:06.07 And you see that the molecule of DNA does behave as a spring, but it is a very non-linear spring.
00:23:13.08 Notice that the data here actually curves in a very, very, very sharp way,
00:23:20.28 indicating that the molecule in fact is not a linear spring.
00:23:26.15 If the molecule would behave as a linear spring it should have in a plot of force versus extension,
00:23:32.09 as this is, right? This plot is force versus extension.
00:23:35.23 We would expect the molecule to behave as the dashed line here in the middle.
00:23:40.11 You see that's actually what would be a Hookean spring, a linear spring.
00:23:44.04 DNA is not a linear spring. It is a highly non-linear spring.
00:23:48.28 Okay, so in fact we studied this for quite awhile, and we were able to show that it is possible
00:24:02.02 to rationalize all these elastic behaviors of the DNA molecule
00:24:05.13 using a model of polymer elasticity called the worm-like chain model.
00:24:10.24 And we derived this expression, shown here, that actually accounts very well
00:24:15.12 for the force need to extend the molecule a given amount
00:24:20.26 in this case, z, when the molecule has a length, a control length L,
00:24:26.25 and the experiment is carried out at the temperature T.
00:24:30.24 This particular model requires the introduction of one parameter that describes the elasticity of the DNA molecule,
00:24:38.10 and that parameter is what we call resistance length.
00:24:42.01 This interpolation formula which I show here in this panel actually turns out to be a very, very good approximation
00:24:50.13 to the exact solution for the force extension behavior of a molecule of DNA.
00:24:55.10 But you may ask, how do we go from pulling a molecule of DNA to actually studying and following reactions in singulo?
00:25:03.22 It turns out that the ability of manipulating one molecule of DNA, as I showed you here,
00:25:13.18 allows us to follow the activity of a number of molecular machines that are actually nucleic acid based molecular machines.
00:25:22.00 And so I want to then tell you a little bit about these molecular machines.
00:25:29.29 Many years ago, some 25 years ago, biologists still thought of the cell as a small bag
00:25:39.07 of a concentrated solution of nucleic acids and proteins
00:25:43.09 that were undergoing second-order reactions.
00:25:46.06 But for the last 25 years or so there has been a paradigmatic shift in the way biologists look at cells.
00:25:53.19 Now in the words of Bruce Alberts in fact in this quite celebrated paper in Cell in 1988,
00:26:02.13 he pointed out that the entire cell really is better described
00:26:08.12 as a "factory that contains an elaborate network of interlocking assembly lines,
00:26:14.01 each of which is composed of a set of large protein machines."
00:26:19.05 And so the idea, the paradigm that has emerged in the last 20 to 25 years or so,
00:26:27.19 is that the cell in fact has a modular factory, a modular architecture.
00:26:35.13 Essentially each central function of the cell and I have labeled here, mentioned several:
00:26:42.03 replication, transcription, translation, translocation, recombination, splicing, protein folding, protein degradation,
00:26:48.20 is really performed by specialized multi-protein assemblies.
00:26:54.01 And this process in units actually typically function as molecular machines.
00:26:59.24 These molecular machines, here is one example.
00:27:05.06 For example the molecular machine responsible for the initiation of transcription in eukaryotic cells,
00:27:10.13 which has more than 60 proteins involved actually,
00:27:15.24 are responsible for one particular function, and they contain various parts with specialized functions.
00:27:23.26 They have dimensions that are in the nanometer scale because they are very, very large.
00:27:30.00 And often they provide a localized environment for reactions to occur.
00:27:34.14 So in fact many of them function as molecular motors, converting chemical energy,
00:27:41.14 which could be in the form of ATP hydrolysis or GTP hydrolysis or in the form of a gradient of protons across the membrane,
00:27:50.10 into mechanical work, producing movement and the generation of force.
00:27:57.12 Well, it turns out that single molecule manipulation methods,
00:28:04.10 as I showed you before, are ideally suited to studying these molecular machines, in particular these molecular motors,
00:28:11.02 because force and displacement, which are the two variables that are most natural
00:28:16.06 for the behavior of these motors, actually are directly observable in single molecule experimental manipulation experiments.
00:28:24.20 So in fact we can now follow a reaction such as this: a single molecule of RNA polymerase as shown here
00:28:36.01 bound to the promoter and transcribing a single molecule of RNA,
00:28:42.23 and synthesizing a single molecule of mRNA and we can actually follow that reaction in real time
00:28:49.00 one molecule at a time. How do we do that?
00:28:53.07 In this case, we would use two optical traps, as indicated here, one trapping one bead
00:28:58.14 that has attached by the back a single molecule of RNA polymerase.
00:29:02.18 This is the eukaryotic RNA polymerase, Pol2.
00:29:06.08 And that molecule is bound already to its promoter, and so we have the rest of the DNA, and the other end of the DNA
00:29:13.16 is attached by a biotin or a digoxigenin or some other chemical moiety to a bead,
00:29:20.16 which is also kept in another, in the second trap.
00:29:23.27 And now we can in fact play tug of war with one of these molecules of RNA polymerase.
00:29:29.24 As we add the NTPs, the nucleoside triphosphates, the molecule will want to actually transcribe the DNA
00:29:38.05 and in order to transcribe the DNA it has to- because it cannot move because it is attached by the back to this bead.
00:29:45.14 In the laboratory frame, it has to pull the DNA through itself and in pulling the DNA through itself,
00:29:51.26 it must pull this second bead out of the trap.
00:29:55.15 And we can follow that movement of the second bead out of the trap,
00:29:58.28 and in this way follow the chemical reaction, the transcription of a single molecule of RNA polymerase
00:30:04.02 in real time one molecule at a time.
00:30:06.14 And in fact you can see here that because the distance between the beads is getting shorter as the molecule transcribes,
00:30:15.12 you can see that... or larger if you actually do the experiment in the opposite geometry...
00:30:22.17 then you can actually follow how the length of the position of the bead changes in the trap as the time passes. Right?
00:30:31.25 So what is that in this case the polymerase is not behaving in a very simple manner.
00:30:37.00 For one, it moves, which corresponds to the regions here in the curve that are black,
00:30:42.22 but then for no apparent reason the molecule stops. It has a pause.
00:30:47.16 It pauses there for awhile, and again it resumes transcription for no apparent reason,
00:30:53.20 a few seconds later and so on and so forth.
00:30:55.14 This kind of dynamics is not accessible in bulk experiments,
00:31:00.21 and it only becomes accessible when you are able to study molecules one at a time.
00:31:05.21 Here is another experiment where we have changed slightly where... It's still starting transcription one molecule at a time,
00:31:13.24 but what we've done is we have now a molecule of DNA attached between two beads,
00:31:19.07 as indicated here in this slide, and one of the beads is kept in an optical trap,
00:31:27.29 and the other bead is as before, atop a micropipette. Right?
00:31:31.22 And what we have now is a molecule of RNA polymerase to which we have attached a tiny little bead on the side
00:31:39.18 and in order for the molecule to actually transcribe the DNA,
00:31:42.12 it must go around, move around the DNA in the spiral of the helix of the DNA.
00:31:47.13 And we will be able to see that in the movie because the bead is large enough
00:31:52.24 that it allows us to follow the actual movement around the DNA as the RNA polymerase transcribes the DNA, as you can see there.
00:32:02.29 Again, this type of experiments allows you to follow the dynamics of the reaction
00:32:10.06 not just the average behavior of RNA polymerase molecules,
00:32:13.05 but how individual molecules behave one time and the following time, and the following second and so on and so forth.
00:32:21.10 Another experiment that we can do is we can actually follow a whole ribosome,
00:32:26.11 as you know this is a very complex machine made out of more than 50 proteins
00:32:31.07 and two different types of RNA, but we can actually follow this process in real time.
00:32:40.11 Right? And so in this case the experiment is different.
00:32:45.05 We put the ribosome at the base of this RNA hairpin, which is going to be translated by the ribosome.
00:32:51.02 And then we have grabbed the two ends of the RNA hairpin.
00:32:57.28 And we have one attached to a bead in the pipette and the other attached to the bead in the trap as before.
00:33:04.14 So now what is going to happen is this. We add all the initiation factors, the tRNAs and the elongation factors,
00:33:12.29 and then this ribosome is going to start translating this RNA.
00:33:19.27 As translation occurs, the ribosome has to open the hairpin here.
00:33:23.12 Right? And as the hairpin opens and is being translated,
00:33:27.06 essentially the RNA molecule, the arms of the RNA molecule, are getting longer and longer, which means that the beads
00:33:36.11 distance actually gets separated. So we can actually follow the reaction
00:33:42.02 in this case by the beads moving apart as a function of time
00:33:47.05 as the ribosome actually enters the hairpin and opens the hairpin.
00:33:52.09 And what you will see here is in fact the translation by a single ribosome.
00:33:58.26 The dashed lines in this plot are separated by three basepairs, and perhaps not surprisingly,
00:34:05.06 but remember this is one single ribosome connecting one single molecule of RNA,
00:34:10.05 what we see is that the ribosome is in fact moving by one codon at a time.
00:34:18.13 3 basepairs, 3 basepairs, 3 basepairs every time.
00:34:29.19 Note that at the end there is a big jump, but that big jump is because what is left of the hairpin is so small
00:34:35.19 that it is quite unstable at the tension that we are applying at the ends of the RNA, and the RNA just spontaneously opens.
00:34:43.16 Okay, so we can also study the ribosome as a molecular motor.
00:34:51.18 because obviously the ribosome in order to move along the mRNA has to be converting part of the chemical energy
00:34:57.27 of the reaction that it is catalyzing, the peptidyl transfer reaction of the peptide synthesis
00:35:03.06 into mechanical work and force and so in this case,
00:35:07.12 what we do is we grab the ribosome by the back,
00:35:09.27 as before, and attach it via biotin and a streptavidin linker to the surface of the bead in the pipette.
00:35:19.00 And the other end of the mRNA is grabbed by a piece of DNA forming a DNA/RNA hybrid
00:35:24.25 onto the surface of a bead that is in the trap. And now this is what we call our tug of war geometry.
00:35:30.20 And now when we add all the reactants for translation to occur,
00:35:34.14 or the reaction mixture for the translation reaction to occur,
00:35:38.06 what we see is that the ribosome in order to translate this mRNA
00:35:44.08 will have to pull the bead out of the trap in order to actually move along the mRNA.
00:35:50.01 And in that case you can see as is shown in this movie,
00:35:54.12 you can see that in fact again notice that the dashed lines are separated by, excuse me, by 3 basepairs.
00:36:02.27 And you see that actually the ribosome strictly moves along the mRNA by a codon at a time.
00:36:13.27 And we can follow that in real time. And you can, more importantly,
00:36:18.02 notice that the ribosome spends time at each codon before it actually makes the next translocation.
00:36:24.03 How is this time distributed?
00:36:26.15 What is happening during these times?
00:36:28.22 How sharp the distribution of times, these waiting times, are?
00:36:33.07 How fast the actual translation from one codon to the next codon can occur?
00:36:38.11 How does that depend on the force applied or the tension applied to the RNA?
00:36:43.17 All of these are questions that we can now access because we have a single molecule assay for this experiment.
00:36:50.07 And in fact in this experiment just to finish this first half, I'd like to show you
00:36:58.21 that we can apply increasing tension in this tug of war experiment
00:37:03.23 between the ribosome and the other end of the mRNA,
00:37:07.12 and see how the rate of translation of the ribosome varies with the applied tension, with the load.
00:37:15.25 In other words we can study the ribosome as a motor, and we are able to characterize
00:37:22.07 for the first time how is it that the motor, this motor, the ribosome
00:37:27.15 responds to a mechanical load. And here is the answer.
00:37:32.09 You see that actually the rate of translation is quite sensitive to the force applied, and that
00:37:37.21 the ribosome can work against forces around 14-15 piconewtons
00:37:43.01 but then at that point the rate of translation becomes very, very small.
00:37:46.14 Okay, so this is an example or a group of examples of how you can do single molecule experiments
00:37:54.04 to study complex biochemical reactions.
00:37:58.08 In the second half of this lecture I will now specifically talk about one particular motor that we have studied in great detail,
00:38:08.10 and hopefully that will illustrate the kind of information that it is possible now to obtain
00:38:14.08 using methods of single molecule manipulation.

This Talk

Speaker: Carlos Bustamante

Audience:

Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: September 2010

Watch in:

Spanish

Talk Overview

Dr. Bustamante begins his talk by explaining why one would wish to study biochemical reactions at the level of single molecules. He explains that many processes within the cell are carried out by very few molecules. By studying single molecules, it is possible to obtain details about the mechanism of a reaction that cannot be ascertained by studying a population of molecules.

Bustamante goes on to describe the technique of optical tweezers and how it can be used to manipulate single molecules. His lab has successfully used this method to follow DNA transcription one molecule at a time and RNA translation one codon at a time. In both cases, single molecule studies provided detailed information about complex biochemical processes.

Speaker Bio

Carlos Bustamante

Dr. Bustamante received his B.S. in biology from Cayetano Heredia Universtiy in Lima, Peru, and his M.S. in biochemistry from San Marcos University, also in Lima. He then moved to the University of California, Berkeley where here completed his Ph.D. in biophysics. Currently, Dr. Bustamante is a Howard Hughes Medical Institute Investigator and a Professor… Continue Reading