WEBVTT 00:00:00.500 --> 00:00:02.830 The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.030 continue to offer high-quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 from hundreds of MIT courses, visit Mit OpenCourseWare 00:00:17.610 --> 00:00:18.520 at ocw.mit.edu. 00:00:25.980 --> 00:00:28.410 ELIZABETH NOLAN: So where we left off last time, 00:00:28.410 --> 00:00:30.660 we were talking about using antibiotics 00:00:30.660 --> 00:00:33.720 as tools to study the ribosome. 00:00:33.720 --> 00:00:37.770 And recall that antibiotics have many different structures, 00:00:37.770 --> 00:00:40.440 can bind to the ribosome at different places. 00:00:40.440 --> 00:00:46.560 And we closed with talking about this antibiotic, puromycin, 00:00:46.560 --> 00:00:49.740 that can bind to the A-site and cause chain termination, 00:00:49.740 --> 00:00:51.870 and also molecules that are derivatives 00:00:51.870 --> 00:00:57.270 of puromycin, such as that more elaborate one with a C75 there. 00:00:57.270 --> 00:01:00.540 And so the example of a system where 00:01:00.540 --> 00:01:03.000 puromycin has been employed, and this 00:01:03.000 --> 00:01:05.459 is just one of many, many examples, 00:01:05.459 --> 00:01:08.830 but also gives us a little new information about players 00:01:08.830 --> 00:01:13.900 in translation, involves studies of elongation factor peak, so 00:01:13.900 --> 00:01:15.610 EFP. 00:01:15.610 --> 00:01:18.630 And if you recall, where I closed last time 00:01:18.630 --> 00:01:23.130 was with the comment that this EFP over the years 00:01:23.130 --> 00:01:26.880 was implicated in a variety of cellular processes. 00:01:26.880 --> 00:01:29.980 But its precise function remained unclear. 00:01:29.980 --> 00:01:33.720 And so Rodnina and co-workers conducted 00:01:33.720 --> 00:01:36.120 a series of experiments to ask, what 00:01:36.120 --> 00:01:41.100 is the effect of EFP on peptide bond formation 00:01:41.100 --> 00:01:46.170 when different dipeptides are in the P-site? 00:01:46.170 --> 00:01:46.860 OK? 00:01:46.860 --> 00:01:49.800 And their experiments were motivated by the fact 00:01:49.800 --> 00:01:53.640 that there was some preliminary work out there suggesting 00:01:53.640 --> 00:01:56.760 that EFP accelerates peptide bond formation, 00:01:56.760 --> 00:01:59.500 but really, the details were unclear. 00:01:59.500 --> 00:02:01.450 So we're going to look at the experiment, 00:02:01.450 --> 00:02:03.450 their initial experiment they did, 00:02:03.450 --> 00:02:06.660 which led to some new understanding about how 00:02:06.660 --> 00:02:11.039 EFP affects the translation process. 00:02:11.039 --> 00:02:14.510 So what is it that they want to do in this experiment 00:02:14.510 --> 00:02:17.280 effectively? 00:02:17.280 --> 00:02:26.480 Imagine we have our ribosome, and we have our three sites, 00:02:26.480 --> 00:02:27.630 OK? 00:02:27.630 --> 00:02:30.000 And so what they do in this experiment is 00:02:30.000 --> 00:02:39.380 they have a dipeptide loaded in the P-site, 00:02:39.380 --> 00:02:41.565 OK, where x is some amino acid. 00:02:47.960 --> 00:02:49.990 OK, and then what they want to do 00:02:49.990 --> 00:02:59.860 is have puromycin in the A-site and then effectively monitor 00:02:59.860 --> 00:03:10.310 for peptide bond formation with or without EFP 00:03:10.310 --> 00:03:19.910 added such that the product is effectively 00:03:19.910 --> 00:03:24.000 a tripeptide, where we have fMat, the amino acid 00:03:24.000 --> 00:03:25.940 and puromycin, OK? 00:03:25.940 --> 00:03:28.400 And keep in mind, if this is what's being monitored, 00:03:28.400 --> 00:03:30.350 there needs to be a step to hydrolyze 00:03:30.350 --> 00:03:35.310 this tripeptide off the tRNA that's in the P-site, OK? 00:03:35.310 --> 00:03:37.290 And throughout this work, how they monitored 00:03:37.290 --> 00:03:44.940 this is that they have a radio label on the formal methionine. 00:03:44.940 --> 00:03:48.390 So you can imagine that you can somehow separate and see 00:03:48.390 --> 00:03:53.670 the dipeptide as well as this tripeptide-like molecule 00:03:53.670 --> 00:03:56.790 with the puromycin attached. 00:03:56.790 --> 00:04:02.080 So how to set up an experiment to test this? 00:04:02.080 --> 00:04:04.950 So they do a stop-flow experiment, 00:04:04.950 --> 00:04:08.220 so you heard some more about that method in recitation 00:04:08.220 --> 00:04:09.960 last week. 00:04:09.960 --> 00:04:11.730 And so in thinking about this, we 00:04:11.730 --> 00:04:15.640 need to think about what will be mixed. 00:04:15.640 --> 00:04:18.450 So what are the components of each syringe? 00:04:18.450 --> 00:04:22.180 How will this reaction be quenched? 00:04:22.180 --> 00:04:25.050 And so beginning to think about that, 00:04:25.050 --> 00:04:27.990 the question is, how do we even get the ribosome 00:04:27.990 --> 00:04:30.640 we need to start with in order to see the reaction? 00:04:30.640 --> 00:04:31.140 Right? 00:04:31.140 --> 00:04:34.350 So imagine that the goal is to have 00:04:34.350 --> 00:04:39.540 a pre-translocation ribosome, so effectively that dipeptide 00:04:39.540 --> 00:04:42.780 is in the P-site, and the A-site's empty. 00:04:42.780 --> 00:04:48.090 And then that assembled post-translocation ribosome 00:04:48.090 --> 00:04:50.970 needs to be mixed with puromycin such 00:04:50.970 --> 00:04:53.820 that puromycin can enter the A-site and peptide bond 00:04:53.820 --> 00:04:55.630 formation can occur. 00:04:55.630 --> 00:04:56.130 OK? 00:04:56.130 --> 00:04:57.588 So there's quite a bit of work that 00:04:57.588 --> 00:05:00.150 needs to happen to even get this experiment set up, 00:05:00.150 --> 00:05:03.240 because somehow that post-translocational ribosome 00:05:03.240 --> 00:05:04.740 needs to be made. 00:05:04.740 --> 00:05:06.960 OK, so if we think about this from the standpoint 00:05:06.960 --> 00:05:11.940 of the experiment and using the stop-flow to rapidly mix, 00:05:11.940 --> 00:05:22.470 we have syringe 1, and we have syringe 2, 00:05:22.470 --> 00:05:24.850 and we have our mixer. 00:05:24.850 --> 00:05:27.100 OK, so what are we going to put in syringe 1? 00:05:34.170 --> 00:05:45.670 OK, so here, we're going to have the post-translocation 00:05:45.670 --> 00:05:46.360 ribosome. 00:05:57.560 --> 00:06:06.280 The A-site is empty, and the P-site 00:06:06.280 --> 00:06:10.260 holds the dipeptide attached to the tRNA. 00:06:20.270 --> 00:06:27.860 And then in syringe 2, we're going to have puromycin here. 00:06:27.860 --> 00:06:31.080 OK, so before we get to EFP, I'm thinking 00:06:31.080 --> 00:06:33.510 about how we're going to look at that in this reaction 00:06:33.510 --> 00:06:34.740 and what it does. 00:06:34.740 --> 00:06:37.020 How are we going to get here? 00:06:37.020 --> 00:06:40.380 So what needs to be done to get this post-translocational 00:06:40.380 --> 00:06:41.453 ribosome? 00:06:47.740 --> 00:06:49.840 Is it in the sigma catalog? 00:06:49.840 --> 00:06:50.550 Bio rad? 00:06:56.340 --> 00:06:57.390 No way! 00:06:57.390 --> 00:06:59.580 And even if it were, you would be broke needing 00:06:59.580 --> 00:07:01.860 to purchase enough to do this experiment, right? 00:07:01.860 --> 00:07:04.470 You talked about needing high concentrations in recitation 00:07:04.470 --> 00:07:07.840 last week for these types of experiments. 00:07:07.840 --> 00:07:09.840 So where does this come from? 00:07:09.840 --> 00:07:12.990 What you need to do before even getting this into your syringe 00:07:12.990 --> 00:07:15.480 here, to do a rapid mixing experiment? 00:07:25.460 --> 00:07:28.090 AUDIENCE: You have to isolate it from cells? 00:07:28.090 --> 00:07:32.710 ELIZABETH NOLAN: OK, so what is the likelihood of isolating-- 00:07:32.710 --> 00:07:36.358 well, what's it, what do you need to isolate from cells? 00:07:36.358 --> 00:07:38.858 AUDIENCE: Well, you're going to need to modify it afterwards 00:07:38.858 --> 00:07:40.160 because there'll be all sorts of other things. 00:07:40.160 --> 00:07:41.743 ELIZABETH NOLAN: Right, but what's it? 00:07:41.743 --> 00:07:42.990 AUDIENCE: The ribosome. 00:07:42.990 --> 00:07:44.920 ELIZABETH NOLAN: OK, so we need a ribosome. 00:07:44.920 --> 00:07:45.590 Right? 00:07:45.590 --> 00:07:47.350 What else do we need? 00:07:47.350 --> 00:07:49.270 So we need the ribosome, and we need 00:07:49.270 --> 00:07:51.820 to get this into the P-site. 00:07:51.820 --> 00:07:56.470 So how are we going to get that dipeptidyl tRNA 00:07:56.470 --> 00:07:57.730 into the P-site? 00:07:57.730 --> 00:07:58.900 AUDIENCE: You need an mRNA. 00:07:58.900 --> 00:08:00.460 ELIZABETH NOLAN: We need an mRNA, 00:08:00.460 --> 00:08:05.140 and we're going to design that mRNA based on what amino acids 00:08:05.140 --> 00:08:06.860 we're interested in. 00:08:06.860 --> 00:08:09.370 So we need to come up with an mRNA. 00:08:09.370 --> 00:08:11.290 What else do we need? 00:08:11.290 --> 00:08:13.390 So think back to the whole cycle. 00:08:13.390 --> 00:08:16.060 AUDIENCE: You need EF-Tu, GTP. 00:08:16.060 --> 00:08:20.950 You need everything necessary to form the fMat to x-peptide. 00:08:20.950 --> 00:08:22.600 ELIZABETH NOLAN: Yeah. 00:08:22.600 --> 00:08:24.550 So what does that mean first? 00:08:24.550 --> 00:08:26.110 And when does that bond form? 00:08:26.110 --> 00:08:27.820 That's the next thing, right? 00:08:27.820 --> 00:08:32.230 So can we deliver this species to the P-site, 00:08:32.230 --> 00:08:34.630 based on what we understand about translation 00:08:34.630 --> 00:08:38.370 in the past four or five lectures? 00:08:38.370 --> 00:08:39.159 No. 00:08:39.159 --> 00:08:39.659 Right? 00:08:39.659 --> 00:08:41.970 So first, the initiation complex needs 00:08:41.970 --> 00:08:45.240 to be prepared in lab, which means 00:08:45.240 --> 00:08:50.010 you need initiation factors, a ribosome, mRNA, the initiator 00:08:50.010 --> 00:08:52.050 tRNA. 00:08:52.050 --> 00:08:56.220 And then that initiation complex needs to be purified, 00:08:56.220 --> 00:08:58.620 which is done by a type of sucrose gradient 00:08:58.620 --> 00:09:00.540 centrifugation. 00:09:00.540 --> 00:09:02.160 OK, and then what? 00:09:02.160 --> 00:09:04.050 Once that initiation complex is formed, 00:09:04.050 --> 00:09:07.590 there needs to be a round of elongation, where 00:09:07.590 --> 00:09:10.200 the ternary complex of EF-Tu. 00:09:10.200 --> 00:09:15.570 The amino acid and GTP comes in to deliver that x-tRNA x 00:09:15.570 --> 00:09:20.070 to this A-site, and then have peptide bond formation occur. 00:09:20.070 --> 00:09:21.420 OK? 00:09:21.420 --> 00:09:24.480 And then, we also need the help of EFG 00:09:24.480 --> 00:09:27.420 to move that to the P-site, right? 00:09:27.420 --> 00:09:29.670 So that whole cycle we've talked about 00:09:29.670 --> 00:09:31.290 from a fundamental perspective needs 00:09:31.290 --> 00:09:35.268 to be done at the bench in order to get here. 00:09:35.268 --> 00:09:36.810 So there's a lot of factors that need 00:09:36.810 --> 00:09:39.270 to be purified and obtained, quite 00:09:39.270 --> 00:09:42.795 a bit of effort to just even set this experiment up. 00:09:42.795 --> 00:09:44.100 OK? 00:09:44.100 --> 00:09:48.600 So always think about where these things come from. 00:09:48.600 --> 00:09:50.040 So we have this. 00:09:50.040 --> 00:09:52.800 We have puromycin, right? 00:09:52.800 --> 00:09:58.800 And then we want to look at the effective EFP. 00:09:58.800 --> 00:10:02.760 So the idea is, are there differences 00:10:02.760 --> 00:10:04.530 in peptide bond formation? 00:10:04.530 --> 00:10:07.050 Is it accelerated in the presence of EFP, 00:10:07.050 --> 00:10:10.710 as how some of this preliminary data indicated? 00:10:10.710 --> 00:10:13.350 And if so, is that for all amino acids? 00:10:13.350 --> 00:10:17.100 Or is it specific for certain amino acids, right? 00:10:17.100 --> 00:10:19.860 So we need to include EFP. 00:10:19.860 --> 00:10:23.573 And in these experiments, it was either omitted or included 00:10:23.573 --> 00:10:24.240 in each syringe. 00:10:35.660 --> 00:10:37.430 And something just to think about when 00:10:37.430 --> 00:10:39.530 thinking about these rapid mixing experiments 00:10:39.530 --> 00:10:41.840 is what happens in the mixer, right? 00:10:41.840 --> 00:10:44.030 If you're having the same volume, which 00:10:44.030 --> 00:10:46.790 is the case coming from syringe 1 and 2, 00:10:46.790 --> 00:10:48.710 you're going to have a dilution in here 00:10:48.710 --> 00:10:50.360 of all of the components. 00:10:50.360 --> 00:10:51.830 Right? 00:10:51.830 --> 00:10:55.160 So these are going to be rapidly mixed in the absence 00:10:55.160 --> 00:10:57.890 or presence of EFP. 00:10:57.890 --> 00:11:01.260 There'll be some time to allow for reaction to occur. 00:11:01.260 --> 00:11:02.930 And then, in this case, the reaction 00:11:02.930 --> 00:11:04.190 is going to be quenched. 00:11:04.190 --> 00:11:05.960 So it's the quench flow-type setup 00:11:05.960 --> 00:11:10.560 that came up in the recitation notes from last week. 00:11:10.560 --> 00:11:12.500 So in this case, we're going to have a syringe 00:11:12.500 --> 00:11:18.440 3 with a quencher. 00:11:18.440 --> 00:11:21.020 And in this particular work, they used base, 00:11:21.020 --> 00:11:22.340 so sometimes it's acid. 00:11:22.340 --> 00:11:24.680 Sometimes it's base. 00:11:24.680 --> 00:11:30.140 And this was a solution of KOH. 00:11:30.140 --> 00:11:38.420 OK, so then after some time, OK, we 00:11:38.420 --> 00:11:39.710 can have the reaction quench. 00:11:47.810 --> 00:11:53.690 OK, and then there'll be some sort of workup and product 00:11:53.690 --> 00:11:54.560 analysis. 00:12:00.050 --> 00:12:01.070 OK? 00:12:01.070 --> 00:12:08.223 So in this case, they chose to hydrolyse the peptidyl tRNA's 00:12:08.223 --> 00:12:09.640 and look at the peptide fragments. 00:12:09.640 --> 00:12:12.050 So you can imagine you need a method that's 00:12:12.050 --> 00:12:15.420 going to separate fMet x, whatever amino acid x 00:12:15.420 --> 00:12:18.550 is, from that product there. 00:12:18.550 --> 00:12:20.420 And then the radio label on the fMat 00:12:20.420 --> 00:12:21.860 is used for quantification. 00:12:25.450 --> 00:12:28.555 So what happens here? 00:12:31.230 --> 00:12:33.610 And I'll just give a summary, and then we'll 00:12:33.610 --> 00:12:35.090 look at it in more detail. 00:12:35.090 --> 00:12:38.140 So what they did in these experiments-- 00:12:38.140 --> 00:12:40.780 and recall that JoAnne talked about in recitation 00:12:40.780 --> 00:12:42.850 last week, when doing these kinetic experiments, 00:12:42.850 --> 00:12:44.500 you have to tweak them quite a bit 00:12:44.500 --> 00:12:48.220 to get the exact good conditions to observe 00:12:48.220 --> 00:12:49.630 what you want to see. 00:12:49.630 --> 00:12:50.890 So imagine that happened. 00:12:50.890 --> 00:12:55.060 We have our k observed, and I'm going 00:12:55.060 --> 00:12:56.950 to show these on a log scale. 00:12:56.950 --> 00:13:04.120 So always keep in mind, paying attention to what type of scale 00:13:04.120 --> 00:13:06.860 the axes are in. 00:13:06.860 --> 00:13:09.310 And so what we're going to look at 00:13:09.310 --> 00:13:15.520 is the k observed for formation of this tripeptide, 00:13:15.520 --> 00:13:17.380 depending on amino acid. 00:13:17.380 --> 00:13:21.655 And I'm going to generalize a bunch of the data here, 00:13:21.655 --> 00:13:23.655 and then we'll look at all the individual cases. 00:13:27.140 --> 00:13:28.160 OK? 00:13:28.160 --> 00:13:34.790 So here, we have x does not equal proline OK, 00:13:34.790 --> 00:13:38.480 and here, not colored in, is no EFP. 00:13:43.180 --> 00:13:49.300 And shaded is k observed for the reactions conducted 00:13:49.300 --> 00:13:51.148 in the presence of EFP. 00:13:51.148 --> 00:13:51.648 OK? 00:14:05.220 --> 00:14:08.130 So what was observed in these studies, 00:14:08.130 --> 00:14:13.940 looking at having many different amino acids here? 00:14:16.480 --> 00:14:20.650 With that, many of these amino acids 00:14:20.650 --> 00:14:22.810 showed negligible difference, whether or not 00:14:22.810 --> 00:14:25.940 EFP was included in the reaction. 00:14:25.940 --> 00:14:27.070 OK? 00:14:27.070 --> 00:14:29.590 And we can look at that data in more detail 00:14:29.590 --> 00:14:31.780 from the paper on the slide. 00:14:31.780 --> 00:14:34.570 What was very striking about these initial experiments 00:14:34.570 --> 00:14:39.140 was what happened in this case, when x equals proline here. 00:14:39.140 --> 00:14:43.630 So effectively, what they observed in this case 00:14:43.630 --> 00:14:47.470 was about 90-fold rate acceleration. 00:14:53.790 --> 00:14:59.130 Effectively, if we compare the k observed for peptide bond 00:14:59.130 --> 00:15:03.510 formation in the absence of EFP, we 00:15:03.510 --> 00:15:05.970 see it's significantly diminished 00:15:05.970 --> 00:15:09.510 for proline if EFP isn't there. 00:15:09.510 --> 00:15:12.600 And along those lines, it was known 00:15:12.600 --> 00:15:19.230 before that proline attached to its tRNA 00:15:19.230 --> 00:15:21.360 is a poorly reactive tRNA. 00:15:21.360 --> 00:15:24.330 So different aminoacyl tRNA's react differently 00:15:24.330 --> 00:15:26.130 in the ribosome. 00:15:26.130 --> 00:15:28.450 So there's that layer of complexity 00:15:28.450 --> 00:15:33.000 we haven't really talked about in this class yet here. 00:15:33.000 --> 00:15:38.720 So if we take a look at all these different examples, 00:15:38.720 --> 00:15:41.300 this one is the outlier. 00:15:41.300 --> 00:15:42.600 OK? 00:15:42.600 --> 00:15:49.380 So what these data indicated is that EFP has some special role 00:15:49.380 --> 00:15:54.420 in accelerating peptide bond formation for peptide bonds 00:15:54.420 --> 00:16:00.420 that contain a C-terminal proline residue here for that. 00:16:00.420 --> 00:16:03.390 And so these experiments were just 00:16:03.390 --> 00:16:06.720 a starting point for many additional experiments 00:16:06.720 --> 00:16:11.250 that ended up showing EFP is really critical for helping 00:16:11.250 --> 00:16:15.000 the ribosome translate sequences that have 00:16:15.000 --> 00:16:17.340 consecutive prolines in a row. 00:16:17.340 --> 00:16:22.260 So either three prolines or maybe a PPG sequence here. 00:16:22.260 --> 00:16:24.900 And in the absence of EFP, what can happen 00:16:24.900 --> 00:16:27.000 is that the ribosome stalls. 00:16:27.000 --> 00:16:30.090 So these aminoacyl tRNA's are not very reactive, 00:16:30.090 --> 00:16:32.280 and the ribosome just gets kind of stuck. 00:16:32.280 --> 00:16:36.810 And you can imagine that's not good for the cell. 00:16:36.810 --> 00:16:39.960 And then if we bring these observations back around 00:16:39.960 --> 00:16:42.900 to some of these early works that were suggesting 00:16:42.900 --> 00:16:47.720 EFP has a role in a diversity of different cellular processes, 00:16:47.720 --> 00:16:48.720 what might we ask? 00:16:48.720 --> 00:16:51.570 We might ask, well, where do the sequences 00:16:51.570 --> 00:16:53.730 of multiple prolines come up? 00:16:53.730 --> 00:16:57.720 So what types of proteins have three prolines 00:16:57.720 --> 00:16:59.430 in a row some place in their sequence? 00:16:59.430 --> 00:17:02.070 Or something like PPG. 00:17:02.070 --> 00:17:04.200 And so they took a look at that. 00:17:04.200 --> 00:17:06.150 And if we think about E. coli, there's 00:17:06.150 --> 00:17:09.390 about 4,000 different proteins, and there's 00:17:09.390 --> 00:17:13.800 a subset of around 270 that have these types of sequences 00:17:13.800 --> 00:17:14.730 in them. 00:17:14.730 --> 00:17:18.190 So not hugely common, but they exist. 00:17:18.190 --> 00:17:21.790 And so then ask, what do these proteins do? 00:17:21.790 --> 00:17:22.290 Right? 00:17:22.290 --> 00:17:24.240 Provided a function is known. 00:17:24.240 --> 00:17:29.190 And so what we see is within that subset of about 00:17:29.190 --> 00:17:32.400 270 proteins, there's examples of proteins 00:17:32.400 --> 00:17:36.200 that are involved in regulation, in metabolism, you know, 00:17:36.200 --> 00:17:37.890 important cellular processes. 00:17:37.890 --> 00:17:39.960 So you can begin to understand why 00:17:39.960 --> 00:17:41.370 it might be that this protein got 00:17:41.370 --> 00:17:44.980 implicated in all these different types of phenomena, 00:17:44.980 --> 00:17:45.480 right? 00:17:45.480 --> 00:17:47.460 But in terms of the details, it's 00:17:47.460 --> 00:17:52.320 really back here in terms of how this translation factor is 00:17:52.320 --> 00:17:56.740 helping the ribosome make a certain subset of peptide bonds 00:17:56.740 --> 00:17:57.960 there. 00:17:57.960 --> 00:18:02.460 So if you're curious about this, the paper's really wonderful. 00:18:02.460 --> 00:18:04.770 There's a number of additional interesting experiments 00:18:04.770 --> 00:18:07.080 that are done and additional methods to these kinetics 00:18:07.080 --> 00:18:07.580 there. 00:18:07.580 --> 00:18:12.190 I'm happy to point you in that direction. 00:18:12.190 --> 00:18:15.465 So yes? 00:18:15.465 --> 00:18:18.916 AUDIENCE: Does this rate of the reaction affect upon 00:18:18.916 --> 00:18:19.892 ribosome folding? 00:18:23.310 --> 00:18:24.910 ELIZABETH NOLAN: It could. 00:18:24.910 --> 00:18:26.370 I mean, basically, you're talking 00:18:26.370 --> 00:18:28.980 about what happens as the polypeptide extrudes 00:18:28.980 --> 00:18:30.570 from the ribosome, right? 00:18:30.570 --> 00:18:32.940 And if you're stalled and have some piece 00:18:32.940 --> 00:18:37.160 of this nascent polypeptide on the outside. 00:18:37.160 --> 00:18:39.150 Ribosomes stalling, yeah, what does 00:18:39.150 --> 00:18:41.700 that do in terms of how trigger factor, for instance, 00:18:41.700 --> 00:18:42.870 interacts. 00:18:42.870 --> 00:18:45.270 That's something we'll talk about in the next module, 00:18:45.270 --> 00:18:47.370 and we'll be getting there on Wednesday, 00:18:47.370 --> 00:18:51.090 I hope, if not Friday. 00:18:51.090 --> 00:18:55.230 So with that, we're going to close discussions of module 1 00:18:55.230 --> 00:18:59.730 in the ribosome with looking at some biotechnology 00:18:59.730 --> 00:19:01.860 and thinking about how we can use 00:19:01.860 --> 00:19:04.680 this fundamental understanding of the ribosome 00:19:04.680 --> 00:19:07.110 to do some new things. 00:19:07.110 --> 00:19:11.490 And so we're going to talk about re-engineering translation 00:19:11.490 --> 00:19:16.590 and ways to use this machinery to incorporate 00:19:16.590 --> 00:19:18.690 unnatural amino acids. 00:19:18.690 --> 00:19:21.600 And so to begin thinking about this, 00:19:21.600 --> 00:19:25.140 we can just consider some questions. 00:19:25.140 --> 00:19:28.680 And so many of us in this room are chemists or chemistry 00:19:28.680 --> 00:19:30.450 majors. 00:19:30.450 --> 00:19:33.280 We can think about organic chemistry, so 5.12, 00:19:33.280 --> 00:19:37.620 5.13, and all of the different organic transformations 00:19:37.620 --> 00:19:39.850 that are presented. 00:19:39.850 --> 00:19:42.720 So if we think about all these organic transformations 00:19:42.720 --> 00:19:47.580 and how they're available to synthetic chemists, 00:19:47.580 --> 00:19:49.860 we see a lot of versatility. 00:19:49.860 --> 00:19:53.230 And we can simply ask ourselves, can such versatility 00:19:53.230 --> 00:19:57.850 be achieved for protein modification? 00:19:57.850 --> 00:19:58.900 What is the toolkit? 00:19:58.900 --> 00:20:02.050 How can that toolkit be expanded? 00:20:02.050 --> 00:20:04.870 And then thinking about this further, 00:20:04.870 --> 00:20:07.790 can we use the translation machinery? 00:20:07.790 --> 00:20:12.460 So is it possible to modify the translation machinery 00:20:12.460 --> 00:20:15.820 to allow us to make peptides or proteins that 00:20:15.820 --> 00:20:17.630 have unnatural amino acids? 00:20:17.630 --> 00:20:22.150 So amino acids are moieties that are not the canonical ones. 00:20:22.150 --> 00:20:23.920 And can we do this in cells? 00:20:23.920 --> 00:20:25.600 Can we do this in a test tube? 00:20:25.600 --> 00:20:28.270 And if we can, what does that provide us 00:20:28.270 --> 00:20:32.450 with in terms of possibilities? 00:20:32.450 --> 00:20:35.200 So the answer is yes, and we're going 00:20:35.200 --> 00:20:37.740 to focus on the how and strengths 00:20:37.740 --> 00:20:41.470 and limitations in terms of our discussions of this machinery 00:20:41.470 --> 00:20:42.430 here. 00:20:42.430 --> 00:20:45.580 I also note-- I believe, JoAnne, this will come up. 00:20:45.580 --> 00:20:48.933 Will you be talking about this in the nucleotide parts, too? 00:20:48.933 --> 00:20:50.350 JOANNE STUBBE: If we get that far. 00:20:50.350 --> 00:20:51.850 ELIZABETH NOLAN: If we get that far. 00:20:51.850 --> 00:20:55.150 So in addition to here, this may come up again 00:20:55.150 --> 00:20:58.510 towards the end of the course, as a tool. 00:20:58.510 --> 00:21:03.520 So hopefully we'll get that far, because that's exciting. 00:21:03.520 --> 00:21:07.000 So let's think about re-engineering translation. 00:21:07.000 --> 00:21:08.680 And we can think about two things. 00:21:08.680 --> 00:21:11.780 We can think about the genetic code here, 00:21:11.780 --> 00:21:14.140 and we can think about the ribosome. 00:21:14.140 --> 00:21:18.070 And so I'll just present you with the questions. 00:21:18.070 --> 00:21:21.010 If we consider the genetic code, what 00:21:21.010 --> 00:21:23.200 can be done to this genetic code to change 00:21:23.200 --> 00:21:26.260 an amino acid in a protein? 00:21:26.260 --> 00:21:29.380 And if we think about the ribosome, what 00:21:29.380 --> 00:21:31.970 can be done to the ribosome to change 00:21:31.970 --> 00:21:34.450 an amino acid in a protein? 00:21:34.450 --> 00:21:38.260 And effectively, can we expand the genetic code 00:21:38.260 --> 00:21:41.320 to encode something other than what it's supposed to encode? 00:21:41.320 --> 00:21:47.320 So can this code allow us to encode an unnatural amino acid? 00:21:47.320 --> 00:21:50.200 And from the standpoint of the ribosome, 00:21:50.200 --> 00:21:53.380 is it possible to design new ribosomes? 00:21:53.380 --> 00:21:56.800 So can we make a new ribosome that 00:21:56.800 --> 00:22:00.830 can incorporate unnatural amino acids into proteins? 00:22:00.830 --> 00:22:03.710 So these are separate but related, 00:22:03.710 --> 00:22:09.130 and we're going to first discuss basically reassigning-- 00:22:09.130 --> 00:22:12.220 is it possible to reassign a codon? 00:22:12.220 --> 00:22:15.830 So why would we want to do this? 00:22:15.830 --> 00:22:19.740 And let's think about that for a minute. 00:22:24.010 --> 00:22:28.030 And what do I mean by expanding the genetic code? 00:22:28.030 --> 00:22:32.350 So if we think about the genetic code, 00:22:32.350 --> 00:22:35.800 we all know that it encodes these 20 amino acids building 00:22:35.800 --> 00:22:39.830 blocks, there's the start codons and the stop codon. 00:22:39.830 --> 00:22:42.880 And effectively, the codons are all used up, right? 00:22:42.880 --> 00:22:45.040 There aren't extra codons floating around 00:22:45.040 --> 00:22:50.150 that we could poach and assign to something else here. 00:22:50.150 --> 00:22:54.070 So can we overcome this? 00:22:54.070 --> 00:22:57.970 And why would we want to do that? 00:22:57.970 --> 00:23:01.930 Just broadly, if we think about being able to put something 00:23:01.930 --> 00:23:06.610 other than a natural amino acid in a protein at a specific 00:23:06.610 --> 00:23:09.340 location-- so exactly where we want it-- 00:23:09.340 --> 00:23:14.140 that opens up many possibilities for experiments. 00:23:14.140 --> 00:23:16.090 And we can think about those experiments 00:23:16.090 --> 00:23:20.440 both happening within a cell or outside of a cell. 00:23:20.440 --> 00:23:22.750 And these are experiments that just wouldn't be 00:23:22.750 --> 00:23:25.150 so easy or feasible otherwise. 00:23:25.150 --> 00:23:29.500 So maybe we'd like to study protein structure. 00:23:29.500 --> 00:23:31.150 What could we do? 00:23:31.150 --> 00:23:34.990 So fluorine is used in NMR quite a bit. 00:23:34.990 --> 00:23:36.730 Imagine if you could site-specifically 00:23:36.730 --> 00:23:39.340 label an unnatural amino acid that 00:23:39.340 --> 00:23:42.130 has a CF3 group, for example, and use 00:23:42.130 --> 00:23:44.040 that in your NMR studies. 00:23:44.040 --> 00:23:46.540 So that's something you'll get to think about in the context 00:23:46.540 --> 00:23:49.140 of problem set two. 00:23:49.140 --> 00:23:53.810 Ways to study protein function, protein localization. 00:23:53.810 --> 00:23:56.260 So for instance, instead of attaching 00:23:56.260 --> 00:24:00.490 GFP, which is big, to a protein of interest, 00:24:00.490 --> 00:24:02.260 maybe it's possible to incorporate 00:24:02.260 --> 00:24:04.330 a fluorescent amino acid that lets you 00:24:04.330 --> 00:24:07.000 see that protein in the cell. 00:24:07.000 --> 00:24:09.987 Protein-protein interactions. 00:24:09.987 --> 00:24:12.070 And maybe we'd like to make a new protein that has 00:24:12.070 --> 00:24:14.450 some desired characteristic. 00:24:14.450 --> 00:24:16.510 So there's a lot of possibilities 00:24:16.510 --> 00:24:20.140 to such technology. 00:24:20.140 --> 00:24:24.190 Just to keep in mind, what do many of us do? 00:24:24.190 --> 00:24:28.240 Many of us are familiar with site-directed mutagenesis, 00:24:28.240 --> 00:24:33.270 where we can change an amino acid in a protein. 00:24:33.270 --> 00:24:36.020 And we learn many, many things from this, 00:24:36.020 --> 00:24:39.350 but it is limited to naturally occurring amino acids. 00:24:39.350 --> 00:24:39.850 Right? 00:24:39.850 --> 00:24:44.920 So we'd like something more versatile. 00:24:44.920 --> 00:24:48.070 If we think about strategies also just a little 00:24:48.070 --> 00:24:58.500 bit, backing up here. 00:24:58.500 --> 00:25:01.070 OK, the first thing I'll just point out 00:25:01.070 --> 00:25:12.900 is that how I'm going to divide this, 00:25:12.900 --> 00:25:15.900 just in case this wasn't clear, is considering 00:25:15.900 --> 00:25:22.650 the native ribosome and then considering 00:25:22.650 --> 00:25:23.940 engineered ribosomes. 00:25:29.475 --> 00:25:31.350 And this is where we're going to focus today. 00:25:34.350 --> 00:25:44.880 And if we consider strategies, other strategies 00:25:44.880 --> 00:25:53.610 to incorporate unnatural amino acids, 00:25:53.610 --> 00:26:00.860 and I guess I'll call these standard, 00:26:00.860 --> 00:26:14.690 we can imagine chemical and biosynthetic. 00:26:14.690 --> 00:26:18.710 And I'm not going to go over a plethora of examples 00:26:18.710 --> 00:26:19.580 for either route. 00:26:19.580 --> 00:26:22.430 There'll be some slides included in the posted 00:26:22.430 --> 00:26:26.210 lecture notes that gives examples and pros and cons. 00:26:26.210 --> 00:26:28.460 But one example I will give here is just 00:26:28.460 --> 00:26:32.420 thinking from the standpoint of a chemical modification, what's 00:26:32.420 --> 00:26:38.790 an example and why we might want to do better. 00:26:38.790 --> 00:26:40.550 OK, so this is independent of something 00:26:40.550 --> 00:26:42.410 like site-directed mutagenesis, where you're 00:26:42.410 --> 00:26:45.950 having an organism do the work. 00:26:45.950 --> 00:26:48.080 So if we just consider an example of a chemical 00:26:48.080 --> 00:26:57.710 modification, there's certain amino acid side chains that 00:26:57.710 --> 00:26:59.960 are amenable to modification. 00:26:59.960 --> 00:27:03.320 So imagine you purify a protein, and you 00:27:03.320 --> 00:27:07.670 want to somehow tag that or label it, right? 00:27:07.670 --> 00:27:10.355 One option is to modify cysteine residues. 00:27:17.070 --> 00:27:21.840 And so iodoacetamide and related reagents are commonly employed, 00:27:21.840 --> 00:27:31.035 so imagine that you have some cysteine. 00:27:33.630 --> 00:27:39.670 You can react this with iodoacetamide 00:27:39.670 --> 00:27:42.670 that has some R group, right? 00:27:42.670 --> 00:27:43.270 What happens? 00:28:11.440 --> 00:28:12.470 Here, OK. 00:28:12.470 --> 00:28:15.260 You can get a covalent modification, 00:28:15.260 --> 00:28:26.400 and maybe this is a fluorophore or something else, right? 00:28:26.400 --> 00:28:31.474 So this is terrific, but what are some potential problems? 00:28:31.474 --> 00:28:34.460 AUDIENCE: Sorry, would this be a way 00:28:34.460 --> 00:28:36.380 to modify the amino acid before it's 00:28:36.380 --> 00:28:37.630 incorporated into the protein? 00:28:37.630 --> 00:28:39.130 Or would this be something you would 00:28:39.130 --> 00:28:41.400 do to modify the cysteine in an assembled protein? 00:28:41.400 --> 00:28:43.567 ELIZABETH NOLAN: Yeah, this would be after the fact. 00:28:43.567 --> 00:28:46.320 So imagine you have some protein. 00:28:46.320 --> 00:28:51.300 You've isolated your protein, and you have some cysteine. 00:28:51.300 --> 00:28:52.530 Right, and you'd like-- 00:28:52.530 --> 00:28:54.900 for some reason, you'd like to modify this protein. 00:28:54.900 --> 00:28:58.170 So maybe a fluorophore to see it. 00:28:58.170 --> 00:29:09.420 Maybe you know, a CF3 group for NMR here, 00:29:09.420 --> 00:29:11.310 which then gets to the point, what are 00:29:11.310 --> 00:29:12.725 possible problems with this? 00:29:12.725 --> 00:29:15.225 AUDIENCE: Do you have to use a mild base to be deprotonated, 00:29:15.225 --> 00:29:16.730 or is it maybe deprotonated based 00:29:16.730 --> 00:29:17.980 on where it is in the protein? 00:29:17.980 --> 00:29:21.130 ELIZABETH NOLAN: Yeah, so that gets to an initial issue, which 00:29:21.130 --> 00:29:24.010 is what's required to have this chemistry to happen? 00:29:24.010 --> 00:29:24.510 Right? 00:29:24.510 --> 00:29:27.210 The cysteine needs to be deprotonated. 00:29:27.210 --> 00:29:29.070 So probably the pH of your buffer 00:29:29.070 --> 00:29:31.230 is going to be elevated some. 00:29:31.230 --> 00:29:33.450 Does your protein or enzyme like that or not? 00:29:33.450 --> 00:29:34.750 Maybe, maybe not. 00:29:34.750 --> 00:29:35.380 Yeah? 00:29:35.380 --> 00:29:37.580 AUDIENCE: You can also run into selectivity issues-- 00:29:37.580 --> 00:29:39.640 I mean, having free cysteine residues isn't common, 00:29:39.640 --> 00:29:41.140 but it could be a potential problem. 00:29:41.140 --> 00:29:43.200 ELIZABETH NOLAN: Yeah, so you need-- 00:29:43.200 --> 00:29:45.180 well, it will depend on the protein, right? 00:29:45.180 --> 00:29:47.910 Is the cysteine free or a disulfide? 00:29:47.910 --> 00:29:50.130 Is it a native cysteine, or have you 00:29:50.130 --> 00:29:53.460 done site-directed mutagenesis first to put this cysteine 00:29:53.460 --> 00:29:55.330 in the position you want? 00:29:55.330 --> 00:29:56.490 Right? 00:29:56.490 --> 00:30:00.150 And then what happens if your protein has multiple cysteines 00:30:00.150 --> 00:30:02.160 building on what Rebecca said, and you 00:30:02.160 --> 00:30:07.050 want to have this label at a site-specific location? 00:30:07.050 --> 00:30:07.860 Right? 00:30:07.860 --> 00:30:10.380 What are you going to do about that? 00:30:10.380 --> 00:30:12.550 Are you going to have non-specific labeling? 00:30:12.550 --> 00:30:15.510 Are you going to mutate out the other cysteines? 00:30:15.510 --> 00:30:18.480 If you do that, what could that mean for your protein 00:30:18.480 --> 00:30:19.950 fold or function? 00:30:19.950 --> 00:30:24.360 There's a number of caveats that need to be considered. 00:30:24.360 --> 00:30:28.680 Nonetheless, it's a possibility to do. 00:30:28.680 --> 00:30:31.410 In terms of time, this is a pretty extreme example, 00:30:31.410 --> 00:30:33.390 but I'll just show one example here 00:30:33.390 --> 00:30:36.330 in thinking about this whole process and what you do, 00:30:36.330 --> 00:30:38.970 which also builds upon Rebecca's question. 00:30:38.970 --> 00:30:42.750 So imagine a protein with two subunits. 00:30:42.750 --> 00:30:47.010 And subunit 1 has a cysteine, and subunit 2 doesn't. 00:30:47.010 --> 00:30:49.530 So for some reason, you want to do this labeling. 00:30:49.530 --> 00:30:52.448 This is actually a protein from my group. 00:30:52.448 --> 00:30:54.240 And we wanted to stick a fluorophore on it. 00:30:54.240 --> 00:30:57.360 So we have a cysteine on one of the two subunits. 00:30:57.360 --> 00:31:00.540 You can run this reaction and get this fluorophore modified 00:31:00.540 --> 00:31:02.980 form here. 00:31:02.980 --> 00:31:07.720 And then you can see that's the case, looking at SDS-PAGE. 00:31:07.720 --> 00:31:09.900 So here we're looking at Coomassie stain that 00:31:09.900 --> 00:31:12.510 shows us total protein, and we see 00:31:12.510 --> 00:31:14.670 there's two subunits, 1 and 2. 00:31:14.670 --> 00:31:16.800 So the molecular weights are a little different, 00:31:16.800 --> 00:31:18.940 and we can separate them on this gel. 00:31:18.940 --> 00:31:20.940 And then if we look in the fluorescence channel, 00:31:20.940 --> 00:31:22.470 what do we see? 00:31:22.470 --> 00:31:25.920 We only see fluorescence associated with subunit 1 00:31:25.920 --> 00:31:29.490 and not subunit 2, which tells us our labeling strategy 00:31:29.490 --> 00:31:31.500 has worked well. 00:31:31.500 --> 00:31:35.550 Like, what we're showing in this equation. 00:31:35.550 --> 00:31:38.010 But what's everything that needs to be done? 00:31:38.010 --> 00:31:41.670 Well, we need to overexpress the protein in some organism. 00:31:41.670 --> 00:31:43.500 In this case, E. coli. 00:31:43.500 --> 00:31:45.900 We need to purify the protein. 00:31:45.900 --> 00:31:48.270 And once we have this purified protein in hand, 00:31:48.270 --> 00:31:51.810 we need to do the chemical reaction for the labeling. 00:31:51.810 --> 00:31:56.430 And then we need to purify that product somehow, 00:31:56.430 --> 00:31:59.190 and that's going to depend on the system you're working at. 00:31:59.190 --> 00:32:01.070 And then it needs to be analyzed, right? 00:32:01.070 --> 00:32:03.510 You always want to know what you're working with, right? 00:32:03.510 --> 00:32:06.690 So was this reaction to 100%? 00:32:06.690 --> 00:32:08.650 Did we end up with a mixture? 00:32:08.650 --> 00:32:11.220 If it's a mixture, what to do about that? 00:32:11.220 --> 00:32:13.500 So what does this mean in terms of time? 00:32:13.500 --> 00:32:15.690 And this is not for all cases, OK? 00:32:15.690 --> 00:32:19.440 This is for this exact case involving this protein shown 00:32:19.440 --> 00:32:21.390 as a cartoon here. 00:32:21.390 --> 00:32:24.690 So it takes about six days from start to finish to overexpress 00:32:24.690 --> 00:32:26.760 and purify it. 00:32:26.760 --> 00:32:29.700 Steps 2 to 4, based on the purification, 00:32:29.700 --> 00:32:32.190 we do another four days, right? 00:32:32.190 --> 00:32:34.680 So that's 10 days from start to finish, just 00:32:34.680 --> 00:32:38.140 to get this protein you'd like to use in your experiment. 00:32:38.140 --> 00:32:38.640 Right? 00:32:38.640 --> 00:32:40.800 And you can imagine if somehow a label 00:32:40.800 --> 00:32:44.130 could be put on in vivo, during this initial step 00:32:44.130 --> 00:32:47.670 here, that that would save some time at the end of the day. 00:32:50.290 --> 00:32:55.740 So before moving on to what's done 00:32:55.740 --> 00:32:57.990 for unnatural amino acid incorporation 00:32:57.990 --> 00:33:00.540 by what we'll call the Schultz method out of Professor Peter 00:33:00.540 --> 00:33:04.980 Schultz's group, just to think about biosynthetic methods 00:33:04.980 --> 00:33:06.300 for a minute. 00:33:06.300 --> 00:33:10.350 So some common ones are done for structural studies. 00:33:10.350 --> 00:33:14.250 So for instance, you can imagine feeding an organism something 00:33:14.250 --> 00:33:18.570 like selenocysteine or selenomethionine. 00:33:18.570 --> 00:33:22.370 Another example is labeling nitrogens or carbons 00:33:22.370 --> 00:33:25.250 for NMR, where the organism is fed, 00:33:25.250 --> 00:33:31.220 say, a labeled amino acid, maybe with N15 or C13 there, right? 00:33:31.220 --> 00:33:33.470 So that's just a biosynthetic method, 00:33:33.470 --> 00:33:36.800 where you're changing the growth conditions, rather 00:33:36.800 --> 00:33:41.870 than doing something to manipulate the genetic code 00:33:41.870 --> 00:33:44.510 or the ribosome. 00:33:44.510 --> 00:33:46.800 So what's the conclusion here? 00:33:53.340 --> 00:34:18.719 What we want is we want a method of site-specific incorporation 00:34:18.719 --> 00:34:22.185 of unnatural amino acids in vivo. 00:34:25.170 --> 00:34:37.409 So in a cell and in a desired organism, depending on what 00:34:37.409 --> 00:34:49.420 you want to do with high efficiency and also fidelity, 00:34:49.420 --> 00:34:54.040 so getting back to that idea and before. 00:34:54.040 --> 00:34:57.170 OK, so why do we want to do this in vivo? 00:35:00.410 --> 00:35:03.230 It allows for studies within cells, 00:35:03.230 --> 00:35:06.290 and you also can purify protein from cells, 00:35:06.290 --> 00:35:09.518 so you can do in vitro experiments as well. 00:35:09.518 --> 00:35:11.060 OK, and you can imagine, if you could 00:35:11.060 --> 00:35:14.540 have all of the pieces of this machinery in a cell, 00:35:14.540 --> 00:35:19.310 maybe there's some technical advantage to that. 00:35:19.310 --> 00:35:23.930 So this is what we're going to consider here. 00:35:23.930 --> 00:35:26.120 So to this question, can the ribosome 00:35:26.120 --> 00:35:29.600 incorporate unnatural amino acids into proteins? 00:35:29.600 --> 00:35:33.500 Effectively, what do we need to think about? 00:35:33.500 --> 00:35:37.130 One, we need to think about relaxing 00:35:37.130 --> 00:35:39.890 the substrate specificity of the aminoacyl tRNA 00:35:39.890 --> 00:35:44.150 synthetase to accommodate some unnatural amino acid, right? 00:35:44.150 --> 00:35:46.040 Somehow that unnatural amino acid 00:35:46.040 --> 00:35:48.300 needs to get to the ribosome. 00:35:48.300 --> 00:35:50.990 So if this can be done, and we can 00:35:50.990 --> 00:35:55.070 make a tRNA that has an unnatural amino acid attached 00:35:55.070 --> 00:36:03.710 to it, can this aminoacyl tRNA get to the A-site 00:36:03.710 --> 00:36:05.340 and do the work? 00:36:05.340 --> 00:36:08.390 So this is the method we're going to talk about 00:36:08.390 --> 00:36:11.510 in some detail for the rest of today 00:36:11.510 --> 00:36:14.540 and into Wednesday, this Schultz method. 00:36:14.540 --> 00:36:19.220 So the idea is that there's a tRNA that's dedicated 00:36:19.220 --> 00:36:21.920 for this unnatural amino acid. 00:36:21.920 --> 00:36:25.160 We see this unnatural amino acid shown here, where 00:36:25.160 --> 00:36:26.915 the UAA is indicated by probe. 00:36:29.510 --> 00:36:32.540 We need an aminoacyl tRNA synthetase 00:36:32.540 --> 00:36:35.300 that will take this unnatural amino acid 00:36:35.300 --> 00:36:39.170 and attach it to the three-prime end of the tRNA to give us 00:36:39.170 --> 00:36:42.920 this aminoacylated tRNA. 00:36:42.920 --> 00:36:43.790 And then what? 00:36:43.790 --> 00:36:47.720 Imagine this tRNA can make its way to the ribosome. 00:36:47.720 --> 00:36:48.500 What happens? 00:36:48.500 --> 00:36:53.310 We need a codon for this aminoacyl tRNA. 00:36:53.310 --> 00:36:55.490 It needs to carry the anticodon, and we're 00:36:55.490 --> 00:36:58.190 going to talk about this in some more detail in a minute. 00:36:58.190 --> 00:37:02.030 So we can have a plasmid that has 00:37:02.030 --> 00:37:05.420 the DNA with the gene of interest in it, right? 00:37:05.420 --> 00:37:11.870 This plasmid DNA can be transformed into, say, E. coli 00:37:11.870 --> 00:37:15.050 that has this machinery here. 00:37:15.050 --> 00:37:19.820 We can have transcription to give the mRNA that 00:37:19.820 --> 00:37:24.230 is from this plasmid DNA. 00:37:24.230 --> 00:37:26.600 And then imagine translations such 00:37:26.600 --> 00:37:29.560 that this unnatural amino acid is incorporated. 00:37:35.680 --> 00:37:39.220 So effectively, where we're going 00:37:39.220 --> 00:37:43.270 is that we need a general method. 00:37:43.270 --> 00:37:47.560 We want this method to be broadly useful, 00:37:47.560 --> 00:37:54.010 where we can genetically encode this unnatural amino acid 00:37:54.010 --> 00:37:58.480 and have it incorporated in response to a unique triplet 00:37:58.480 --> 00:38:00.280 codon, here. 00:38:03.220 --> 00:38:09.610 So in thinking about that, what are the pieces that we need? 00:38:43.750 --> 00:38:47.560 And we'll think about E. coli for the moment, 00:38:47.560 --> 00:38:48.970 but this could be other. 00:38:48.970 --> 00:38:51.370 So yeast, mammalian cells, right? 00:38:51.370 --> 00:38:57.060 Let your imagination run wild with this here. 00:38:57.060 --> 00:39:11.750 When the incorporation of the UAA in response 00:39:11.750 --> 00:39:18.020 to a unique triplet codon. 00:39:22.080 --> 00:39:25.230 So if we're going to do this, what do we need? 00:39:32.060 --> 00:39:36.330 OK, effectively, we need some new components of the trans-- 00:39:36.330 --> 00:39:39.480 like protein biosynthetic translation machinery, right? 00:39:39.480 --> 00:39:42.725 So we need to rewind and think about the whole translation 00:39:42.725 --> 00:39:43.225 process. 00:40:01.380 --> 00:40:04.450 OK, so the first order of business 00:40:04.450 --> 00:40:06.790 is that we need a unique codon. 00:40:11.610 --> 00:40:12.110 Right? 00:40:12.110 --> 00:40:19.230 So this only designates or uniquely designates the UAA. 00:40:26.710 --> 00:40:29.930 And so we need to ask, where does this come from? 00:40:29.930 --> 00:40:31.520 Because we just went over the fact 00:40:31.520 --> 00:40:37.790 that the codons are used up for amino acid start and stop. 00:40:37.790 --> 00:40:38.900 We need a new tRNA. 00:40:43.620 --> 00:40:56.460 OK, so this tRNA needs to be specific for the unique codon. 00:41:02.810 --> 00:41:14.990 OK, and we need the corresponding aminoacyl tRNA 00:41:14.990 --> 00:41:18.480 synthetase, right? 00:41:18.480 --> 00:41:25.300 And we need this to load the unnatural amino acid 00:41:25.300 --> 00:41:35.370 onto the unique tRNA here. 00:41:35.370 --> 00:41:40.120 So what is a key feature of all of this? 00:41:40.120 --> 00:41:48.170 A key feature is that if we want to do this in some organism, 00:41:48.170 --> 00:41:51.410 we need this machinery to be orthogonal to the machinery 00:41:51.410 --> 00:41:52.145 in that organism. 00:41:56.450 --> 00:42:00.080 We cannot have cross-reactivity, because then there's not going 00:42:00.080 --> 00:42:03.380 to be any selectivity of this incorporation. 00:42:38.580 --> 00:42:40.200 So no cross-reaction. 00:42:46.060 --> 00:42:51.420 So what do we need to consider, in terms of these? 00:42:51.420 --> 00:42:55.260 We need to think about all of the machinery, right? 00:42:55.260 --> 00:43:00.160 And I just list some considerations here. 00:43:00.160 --> 00:43:04.530 So this new tRNA can only allow for translation 00:43:04.530 --> 00:43:07.230 of the codon for the UAA. 00:43:07.230 --> 00:43:10.890 It can't be a substrate for any of the endogenous aaRS, 00:43:10.890 --> 00:43:13.230 because then it will become loaded, potentially, 00:43:13.230 --> 00:43:14.610 with the wrong amino acid. 00:43:14.610 --> 00:43:18.570 So think back to lectures 2 and 3. 00:43:18.570 --> 00:43:20.700 This new aminoacyl tRNA synthetase 00:43:20.700 --> 00:43:24.690 can only recognize the new tRNA and not endogenous tRNA. 00:43:24.690 --> 00:43:26.220 So cross-reactivity again. 00:43:28.740 --> 00:43:32.370 This unnatural amino acid also can't be a substrate 00:43:32.370 --> 00:43:35.130 for endogenous enzymes. 00:43:35.130 --> 00:43:36.600 And also keep in mind, there needs 00:43:36.600 --> 00:43:39.330 to be some way to get this unnatural amino acid 00:43:39.330 --> 00:43:43.050 into a cell if we want to do this in a cellular context. 00:43:43.050 --> 00:43:47.190 So there's just transport issue that needs to be kept in mind. 00:43:47.190 --> 00:43:51.570 Will this unnatural amino acid get into the cell? 00:43:51.570 --> 00:43:59.100 OK, so what we're going to do is consider these requirements 00:43:59.100 --> 00:44:02.070 and what was done to build up this methodology 00:44:02.070 --> 00:44:04.240 during initial work. 00:44:04.240 --> 00:44:07.980 So the first issue is this unique codon, 00:44:07.980 --> 00:44:09.960 and what is its identity here? 00:44:28.650 --> 00:44:33.860 And so if we consider the 64 codons, 00:44:33.860 --> 00:44:41.400 they're used up with the 20 common amino acids. 00:44:41.400 --> 00:44:47.220 We have the three stop codons and the one start codon. 00:44:50.850 --> 00:44:54.660 And so in thinking about this, can ask, 00:44:54.660 --> 00:44:57.690 do we really need three stop codons? 00:44:57.690 --> 00:45:00.030 We certainly need our start, and we need 00:45:00.030 --> 00:45:02.310 codons for the amino acids. 00:45:02.310 --> 00:45:05.490 But is there some wiggle room here? 00:45:05.490 --> 00:45:14.420 And so in terms of these stop codons, 00:45:14.420 --> 00:45:21.960 we have TAA, TAG, and TGA. 00:45:21.960 --> 00:45:23.165 And these all have names. 00:45:27.200 --> 00:45:31.710 Ochre, amber, and opal. 00:45:31.710 --> 00:45:37.220 OK, and so the idea we're going to see is just the question, 00:45:37.220 --> 00:45:39.860 can we reassign a stop codon? 00:45:39.860 --> 00:45:42.380 And can we reassign a stop codon such 00:45:42.380 --> 00:45:45.530 that it's the codon for the unnatural amino acid? 00:45:48.090 --> 00:45:52.340 And so basically, if we want to reassign a stop codon, 00:45:52.340 --> 00:45:54.440 how do we choose? 00:45:54.440 --> 00:45:55.910 Right? 00:45:55.910 --> 00:45:59.080 So two things to consider. 00:45:59.080 --> 00:46:04.160 One, how frequently is each stop codon used? 00:46:04.160 --> 00:46:06.920 So what do we know about that? 00:46:06.920 --> 00:46:13.340 And then does this stop codon terminate essential genes? 00:46:13.340 --> 00:46:16.850 So we can imagine that if we were to reassign a stop 00:46:16.850 --> 00:46:21.860 codon that's used frequently by E. coli or another host, 00:46:21.860 --> 00:46:24.680 or if we were to reassign a stop codon that's 00:46:24.680 --> 00:46:28.640 important for terminating the synthesis of essential genes, 00:46:28.640 --> 00:46:32.480 in either case, the outcome could be pretty bad, right? 00:46:32.480 --> 00:46:36.710 So what was found in thinking about those issues is 00:46:36.710 --> 00:46:43.070 this amber stop codon, TAG, one, it's the least frequently used. 00:46:51.410 --> 00:47:01.460 And just for an example, about 9% in E. coli and about 23% 00:47:01.460 --> 00:47:05.000 in yeast for terminating genes. 00:47:05.000 --> 00:47:10.190 And additionally, it rarely terminates essential genes. 00:47:23.760 --> 00:47:29.310 OK, so based on this, it was decided 00:47:29.310 --> 00:47:47.220 to reassign TAG as the codon for the unnatural amino acid. 00:47:58.240 --> 00:48:03.270 OK, so we've gotten through to here. 00:48:03.270 --> 00:48:08.210 So the question is now, what about requirements 2 and 3? 00:48:08.210 --> 00:48:08.913 So yeah? 00:48:08.913 --> 00:48:10.080 AUDIENCE: I have a question. 00:48:10.080 --> 00:48:12.520 So it seems interesting to choose a stop codon 00:48:12.520 --> 00:48:14.580 to change because if the stop codon messes up, 00:48:14.580 --> 00:48:17.940 it seems more catastrophic to us all than one 00:48:17.940 --> 00:48:21.046 of the other redundant amino acids. 00:48:21.046 --> 00:48:22.760 Like, why use a stop codon? 00:48:22.760 --> 00:48:24.920 I think it's interesting. 00:48:24.920 --> 00:48:27.230 ELIZABETH NOLAN: Yeah, so there is a risk. 00:48:27.230 --> 00:48:29.400 There's certainly a risk, right? 00:48:29.400 --> 00:48:31.980 But these considerations were made 00:48:31.980 --> 00:48:35.280 to try to diminish that risk. 00:48:35.280 --> 00:48:35.970 Right? 00:48:35.970 --> 00:48:41.220 So you could make the argument that maybe all of these stop 00:48:41.220 --> 00:48:43.230 codons aren't essential, right? 00:48:43.230 --> 00:48:44.730 And what is more deleterious? 00:48:44.730 --> 00:48:49.590 Will it be to try to use a stop codon that's infrequently used, 00:48:49.590 --> 00:48:54.600 or to reassign a codon that's for an amino acid that comes up 00:48:54.600 --> 00:48:58.290 in many, many different proteins in the cellular pool? 00:48:58.290 --> 00:48:58.890 Right? 00:48:58.890 --> 00:49:03.490 So there's a judgment call there. 00:49:03.490 --> 00:49:07.980 But if we consider in E. coli, this TAG stop is for about 9% 00:49:07.980 --> 00:49:09.120 of proteins. 00:49:09.120 --> 00:49:13.620 How does that compare to, say, reassigning one of the codons 00:49:13.620 --> 00:49:16.650 to incorporate a lysine or a valine. 00:49:16.650 --> 00:49:18.690 I don't know, but that was just want 00:49:18.690 --> 00:49:21.480 to think about, how frequently is that codon used? 00:49:21.480 --> 00:49:23.490 Because certainly there's different codon usage 00:49:23.490 --> 00:49:24.750 in different organisms. 00:49:24.750 --> 00:49:25.958 Do you have something to say? 00:49:25.958 --> 00:49:27.458 JOANNE STUBBE: So it depends on what 00:49:27.458 --> 00:49:29.620 you want to put the unnatural amino acid in for. 00:49:29.620 --> 00:49:33.460 So if you want it in endogenous levels, it could be a problem. 00:49:33.460 --> 00:49:35.210 But if you're overproducing your protein-- 00:49:35.210 --> 00:49:36.895 ELIZABETH NOLAN: Yeah, it may not be a problem. 00:49:36.895 --> 00:49:39.353 JOANNE STUBBE: Then it's not a problem, because you induce, 00:49:39.353 --> 00:49:41.057 and then you flood it with that and you 00:49:41.057 --> 00:49:42.940 get high levels of cooperation. 00:49:42.940 --> 00:49:45.428 So it depends on what your purpose is. 00:49:45.428 --> 00:49:47.220 ELIZABETH NOLAN: Yeah, so is JoAnne's point 00:49:47.220 --> 00:49:49.290 clear to everyone? 00:49:49.290 --> 00:49:55.260 So you could imagine expressing at an endogenous level, right? 00:49:55.260 --> 00:49:58.410 Or you could imagine causing the cell 00:49:58.410 --> 00:50:01.047 to overexpress the proteins, like off a plasmid, 00:50:01.047 --> 00:50:02.880 like what many of you have done in lab class 00:50:02.880 --> 00:50:05.310 or maybe in research there for that. 00:50:07.830 --> 00:50:10.340 Are there many examples of reassigning a different one? 00:50:10.340 --> 00:50:11.430 JOANNE STUBBE: I think it's really tough. 00:50:11.430 --> 00:50:12.888 I mean, inside the cell, you really 00:50:12.888 --> 00:50:15.840 do have problems if you don't re-engineer, 00:50:15.840 --> 00:50:17.820 because you get truncations also. 00:50:17.820 --> 00:50:20.070 ELIZABETH NOLAN: Yeah, we're going to talk about that. 00:50:20.070 --> 00:50:22.500 So there is a big problem about the stop 00:50:22.500 --> 00:50:24.210 that we're going to talk about, once we 00:50:24.210 --> 00:50:28.740 get to how we have this done, which is premature termination. 00:50:28.740 --> 00:50:32.435 AUDIENCE: I'm kind of confused more or less at that point, 00:50:32.435 --> 00:50:35.970 because stop codon, we're just using because it's not 00:50:35.970 --> 00:50:38.030 currently-- it doesn't go for anything, 00:50:38.030 --> 00:50:43.530 it just ends, like, endogenous sequences that 00:50:43.530 --> 00:50:46.350 are not the UAA so if we replace it, we might not get those. 00:50:46.350 --> 00:50:49.970 But we'll get the one that we're trying to synthesize. 00:50:49.970 --> 00:50:52.750 ELIZABETH NOLAN: Right, so we need a codon 00:50:52.750 --> 00:50:54.640 for the unnatural amino acid. 00:50:54.640 --> 00:50:56.320 And right now, we're limiting our space 00:50:56.320 --> 00:50:58.870 to triplet codons, which is what was initially 00:50:58.870 --> 00:51:02.020 done when this type of methodology was developed. 00:51:02.020 --> 00:51:08.398 So the question is, we have four options in terms of bases. 00:51:08.398 --> 00:51:09.940 AUDIENCE: That are not coding, right? 00:51:09.940 --> 00:51:12.232 ELIZABETH NOLAN: No, no, in terms of our codons, right? 00:51:12.232 --> 00:51:13.030 So three, right? 00:51:13.030 --> 00:51:15.010 Four, three, so there's 64 codons, 00:51:15.010 --> 00:51:16.395 and they're all used up. 00:51:16.395 --> 00:51:17.020 AUDIENCE: Yeah. 00:51:17.020 --> 00:51:18.890 ELIZABETH NOLAN: Right, so there's not some extra. 00:51:18.890 --> 00:51:19.515 AUDIENCE: Yeah. 00:51:19.515 --> 00:51:21.780 ELIZABETH NOLAN: So then what can be reassigned? 00:51:21.780 --> 00:51:23.560 AUDIENCE: These stop-- so the stop. 00:51:23.560 --> 00:51:24.310 ELIZABETH NOLAN: Well, yeah, well, we 00:51:24.310 --> 00:51:25.540 can't reassign the start. 00:51:25.540 --> 00:51:27.540 Then there's no proteins, right? 00:51:27.540 --> 00:51:29.500 There's only one start codon. 00:51:29.500 --> 00:51:34.370 So the thinking was, is a stop codon dispensable? 00:51:34.370 --> 00:51:34.870 Right? 00:51:34.870 --> 00:51:36.610 And then a decision was made, based 00:51:36.610 --> 00:51:40.750 on basically the frequency of use of the stop codon 00:51:40.750 --> 00:51:43.030 and whether or not the stop codon terminates 00:51:43.030 --> 00:51:44.470 essential genes. 00:51:44.470 --> 00:51:46.000 Is this something foolproof? 00:51:46.000 --> 00:51:48.130 No, there's major problems in terms 00:51:48.130 --> 00:51:49.990 of yields that come up as a result. 00:51:49.990 --> 00:51:52.090 And we'll see that on Wednesday, right? 00:51:52.090 --> 00:51:55.810 But you need a starting point to get a method underway. 00:51:55.810 --> 00:51:58.690 So where will we begin tomorrow is 00:51:58.690 --> 00:52:02.500 talking about where this tRNA and the aaRS 00:52:02.500 --> 00:52:04.980 come from to do this.