WEBVTT 00:00:00.500 --> 00:00:02.940 The following content is provided under a Creative 00:00:02.940 --> 00:00:04.270 Commons license. 00:00:04.270 --> 00:00:06.780 Your support will help MIT OpenCourseWare 00:00:06.780 --> 00:00:11.030 continue to offer high-quality educational resources for free. 00:00:11.030 --> 00:00:13.770 To make a donation or view additional materials 00:00:13.770 --> 00:00:17.250 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.250 --> 00:00:20.412 at ocw.mit.edu. 00:00:20.412 --> 00:00:21.870 ELIZABETH NOLAN: Today's recitation 00:00:21.870 --> 00:00:26.910 will all stem from this reading by Youngman and Green 00:00:26.910 --> 00:00:28.650 on ribosome purification. 00:00:28.650 --> 00:00:31.606 But also, we posted an optional-- well, 00:00:31.606 --> 00:00:33.480 not really optional but an additional reading 00:00:33.480 --> 00:00:38.100 that's a review that talks about purifying macromolecular 00:00:38.100 --> 00:00:39.342 machines from the source. 00:00:39.342 --> 00:00:40.800 And I guess one thing I'd just like 00:00:40.800 --> 00:00:43.100 to say about this review, something 00:00:43.100 --> 00:00:46.530 I like is figure three because it indicates 00:00:46.530 --> 00:00:49.140 many, many different types of methods that can be used 00:00:49.140 --> 00:00:51.812 to purify some biomolecule. 00:00:51.812 --> 00:00:55.030 And I think often it's easy just to think all the time, 00:00:55.030 --> 00:00:56.450 well, let's just use a tag. 00:00:56.450 --> 00:00:58.970 And tags have enabled many things, 00:00:58.970 --> 00:01:00.780 but there's also many possibilities 00:01:00.780 --> 00:01:03.150 out there and decades worth of work 00:01:03.150 --> 00:01:05.310 before tags for how to purify proteins. 00:01:05.310 --> 00:01:07.680 And in some instances, you might not want to use a tag 00:01:07.680 --> 00:01:10.630 and that's discussed to some degree here. 00:01:10.630 --> 00:01:12.780 So I guess I'm curious. 00:01:12.780 --> 00:01:15.690 What did you all think about this week's paper? 00:01:15.690 --> 00:01:16.830 Did you like it? 00:01:16.830 --> 00:01:17.760 Did you not like it? 00:01:17.760 --> 00:01:19.710 Was it easy to hard read? 00:01:27.300 --> 00:01:28.990 Did you read the paper? 00:01:35.295 --> 00:01:37.235 AUDIENCE: It wasn't too bad. 00:01:37.235 --> 00:01:38.036 I've purified-- 00:01:38.036 --> 00:01:39.660 JOANNE STUBBE: You need to speak louder 00:01:39.660 --> 00:01:40.630 because I can't hear you. 00:01:40.630 --> 00:01:41.796 AUDIENCE: It wasn't too bad. 00:01:41.796 --> 00:01:43.898 I've purified proteins before, so I 00:01:43.898 --> 00:01:47.090 felt like I could follow along. 00:01:47.090 --> 00:01:49.820 ELIZABETH NOLAN: So not too bad means 00:01:49.820 --> 00:01:53.620 it was easy to understand and follow the text there. 00:01:53.620 --> 00:01:57.420 Yeah, so compared to the reading for recitations two and three, 00:01:57.420 --> 00:02:00.420 this one was probably easier to work with, 00:02:00.420 --> 00:02:03.780 but there's a lot of details in this one too there. 00:02:03.780 --> 00:02:06.870 One thing, I guess, I like this paper from the standpoint of it 00:02:06.870 --> 00:02:10.710 being a methods paper, is the amount of detail 00:02:10.710 --> 00:02:14.640 they give with how they did this purification and things that 00:02:14.640 --> 00:02:16.230 didn't work, right? 00:02:16.230 --> 00:02:19.980 So often, sharing those details with your readers 00:02:19.980 --> 00:02:23.480 can make such difference for an experimentalist 00:02:23.480 --> 00:02:26.220 in another lab in another part of the world, right? 00:02:26.220 --> 00:02:30.510 And I think, from this, one with biochemistry training 00:02:30.510 --> 00:02:34.420 could go into the lab and reproduce their purification 00:02:34.420 --> 00:02:34.920 there. 00:02:34.920 --> 00:02:38.340 So it's a good example in terms of what details to include 00:02:38.340 --> 00:02:41.810 and what types of pitfalls to include. 00:02:41.810 --> 00:02:44.970 So how many of you have done a protein purification, 00:02:44.970 --> 00:02:48.312 either in a lab class or in your research? 00:02:48.312 --> 00:02:49.280 OK. 00:02:49.280 --> 00:02:52.920 And so what type of purification did you use? 00:02:52.920 --> 00:02:56.647 AUDIENCE: We used a histamine-tag with nickel. 00:02:56.647 --> 00:02:57.480 ELIZABETH NOLAN: OK. 00:02:57.480 --> 00:03:00.720 So you used a His6-tag, probably a polyhistamine tag 00:03:00.720 --> 00:03:02.370 in a nickel NTA column. 00:03:02.370 --> 00:03:05.220 For everyone else, has it been that type of methodology 00:03:05.220 --> 00:03:06.450 or another methodology? 00:03:06.450 --> 00:03:07.845 AUDIENCE: I used the same one. 00:03:07.845 --> 00:03:09.050 ELIZABETH NOLAN: OK. 00:03:09.050 --> 00:03:09.550 OK. 00:03:09.550 --> 00:03:12.750 So would someone like to kind of comment 00:03:12.750 --> 00:03:15.890 on the basics of affinity tag purification? 00:03:15.890 --> 00:03:16.950 So how does this work? 00:03:21.530 --> 00:03:23.210 And why did you do it? 00:03:23.210 --> 00:03:25.400 So what are the advantages? 00:03:31.601 --> 00:03:33.300 AUDIENCE: So you have a light chain 00:03:33.300 --> 00:03:36.950 of histamines on your protein, and you 00:03:36.950 --> 00:03:40.680 use nickel, which is a metal that chelates 00:03:40.680 --> 00:03:42.640 to histamine very well. 00:03:47.447 --> 00:03:48.030 Is that right? 00:03:48.030 --> 00:03:48.540 ELIZABETH NOLAN: Yeah. 00:03:48.540 --> 00:03:50.164 The nickel's bound to something though. 00:03:50.164 --> 00:03:52.125 You have the NTA ligand on the resin. 00:03:57.157 --> 00:03:58.698 AUDIENCE: I did this over a year ago. 00:03:58.698 --> 00:03:59.670 Sorry. 00:03:59.670 --> 00:04:02.690 AUDIENCE: So if you have whatever 00:04:02.690 --> 00:04:06.810 the type is, the [INAUDIBLE] bound to some solid substrate. 00:04:06.810 --> 00:04:09.300 And then you can elute everything 00:04:09.300 --> 00:04:11.300 that's not bond to that. 00:04:11.300 --> 00:04:12.690 Elute what it does bind. 00:04:12.690 --> 00:04:13.857 So you just tag the protein. 00:04:13.857 --> 00:04:15.273 It's bound to a high concentration 00:04:15.273 --> 00:04:16.107 of the free ligand. 00:04:19.110 --> 00:04:20.860 ELIZABETH NOLAN: Or right, to push it off. 00:04:20.860 --> 00:04:25.390 So the idea is that you have your biomolecule of interest, 00:04:25.390 --> 00:04:28.100 whether that be a protein, which is what we're all 00:04:28.100 --> 00:04:31.060 most familiar with, or the ribosome, 00:04:31.060 --> 00:04:32.620 and then you attach a tag. 00:04:32.620 --> 00:04:34.900 And that tag can be any number of things, right? 00:04:34.900 --> 00:04:38.050 So in this paper, we saw they used a stem loop structure 00:04:38.050 --> 00:04:41.505 incorporated into the 23s rRNA. 00:04:41.505 --> 00:04:42.880 And the idea is that you're going 00:04:42.880 --> 00:04:47.980 to use that tag to separate your biomolecule of interest 00:04:47.980 --> 00:04:51.610 from the complexity of the cellular environment 00:04:51.610 --> 00:04:53.840 there, so all of the other proteins. 00:04:53.840 --> 00:04:56.530 And so you have some bead or resin 00:04:56.530 --> 00:04:58.130 that this tag can bind to. 00:04:58.130 --> 00:04:59.680 So in the case of the nickel column, 00:04:59.680 --> 00:05:04.370 the His6-tag will bind to the nickel NTA on the resin. 00:05:04.370 --> 00:05:07.330 And then you can wash away other components. 00:05:07.330 --> 00:05:11.650 And then you devise some method to elute the protein 00:05:11.650 --> 00:05:14.380 you hope to have trapped there. 00:05:14.380 --> 00:05:17.740 So what are some advantages of using an affinity tag, 00:05:17.740 --> 00:05:19.210 just thinking about this generally 00:05:19.210 --> 00:05:20.620 before we delve into the paper? 00:05:25.380 --> 00:05:27.417 AUDIENCE: Easy to install. 00:05:27.417 --> 00:05:28.250 ELIZABETH NOLAN: OK. 00:05:28.250 --> 00:05:31.803 So what do you mean by easy to install? 00:05:31.803 --> 00:05:33.775 AUDIENCE: If you wanted to just encode 00:05:33.775 --> 00:05:40.184 a 6 His-tag at the terminus of your target protein, 00:05:40.184 --> 00:05:42.170 I don't know if you can say it's trivial. 00:05:42.170 --> 00:05:43.089 It's easy. 00:05:43.089 --> 00:05:44.630 ELIZABETH NOLAN: I'd agree it's easy. 00:05:44.630 --> 00:05:46.600 There's many plasmids available that you 00:05:46.600 --> 00:05:50.020 can insert your gene of interest into in order to have 00:05:50.020 --> 00:05:51.820 this tag genetically encoded. 00:05:51.820 --> 00:05:54.610 So when you express the protein, the tag's there. 00:05:54.610 --> 00:05:58.440 So beyond that, from the standpoint of purification-- 00:05:58.440 --> 00:06:01.280 AUDIENCE: It's more specific. 00:06:01.280 --> 00:06:02.280 ELIZABETH NOLAN: Pardon? 00:06:02.280 --> 00:06:03.400 AUDIENCE: It's more specific. 00:06:03.400 --> 00:06:04.733 ELIZABETH NOLAN: More specific-- 00:06:04.733 --> 00:06:08.960 AUDIENCE: Pure proteins as opposed to various charges. 00:06:08.960 --> 00:06:11.050 AUDIENCE: It simplifies the purification. 00:06:11.050 --> 00:06:13.976 Instead of doing size exclusion and ion exchange 00:06:13.976 --> 00:06:15.850 that is much different. 00:06:15.850 --> 00:06:18.790 ELIZABETH NOLAN: So the hope is it simplifies the purification 00:06:18.790 --> 00:06:20.890 because you have some way initially 00:06:20.890 --> 00:06:26.620 to pull your protein of interest out of your cell lysate there. 00:06:26.620 --> 00:06:30.040 So that can be a big help. 00:06:30.040 --> 00:06:32.790 What are some potential disadvantages of using a tag? 00:06:32.790 --> 00:06:36.086 So have any of you run into trouble with a tag in the lab? 00:06:36.086 --> 00:06:38.882 AUDIENCE: Having a tag can highly deform your protein 00:06:38.882 --> 00:06:39.724 and change it. 00:06:39.724 --> 00:06:40.640 ELIZABETH NOLAN: Yeah. 00:06:40.640 --> 00:06:43.120 So it might change your protein or deform it. 00:06:43.120 --> 00:06:45.262 What do you mean by "deform?" 00:06:45.262 --> 00:06:47.617 AUDIENCE: It could just cause a conformation change 00:06:47.617 --> 00:06:51.251 or the tag could make it localize somewhere else based 00:06:51.251 --> 00:06:52.794 on size. 00:06:52.794 --> 00:06:53.710 ELIZABETH NOLAN: Yeah. 00:06:53.710 --> 00:06:57.520 So that's an example, say if you were doing a study in cells, 00:06:57.520 --> 00:06:58.870 say, rather than a purification. 00:06:58.870 --> 00:07:00.286 But maybe you tag a protein and it 00:07:00.286 --> 00:07:03.250 goes somewhere other than it would go untagged right? 00:07:03.250 --> 00:07:05.920 And that will affect your observations 00:07:05.920 --> 00:07:08.050 and your data there. 00:07:08.050 --> 00:07:09.670 And it might alter the conformation. 00:07:09.670 --> 00:07:11.650 So it might affect the folding, right? 00:07:11.650 --> 00:07:14.660 It might affect the oligomerization. 00:07:14.660 --> 00:07:17.020 His-tags bind metal ions. 00:07:17.020 --> 00:07:22.140 So is that a factor to consider there? 00:07:22.140 --> 00:07:26.910 If you have an enzyme, will the tag affect activity, right? 00:07:26.910 --> 00:07:29.020 And these things can be a positive or a negative. 00:07:29.020 --> 00:07:32.500 Sometimes the tag is helpful in these regards. 00:07:32.500 --> 00:07:36.670 You can't get soluble protein without the tag, right? 00:07:36.670 --> 00:07:38.800 And sometimes the reverse. 00:07:38.800 --> 00:07:42.190 You decide to express your protein or biomolecule 00:07:42.190 --> 00:07:45.520 with a tag and you find out you get an aggregate, something 00:07:45.520 --> 00:07:47.360 that the protein shouldn't be. 00:07:47.360 --> 00:07:51.580 So these are just things to keep in mind when designing a fusion 00:07:51.580 --> 00:07:54.040 protein and thinking about how you're 00:07:54.040 --> 00:07:58.450 going to use an affinity tag to express your protein there 00:07:58.450 --> 00:08:00.820 and purify your protein. 00:08:00.820 --> 00:08:03.280 So there's pluses and minuses, right? 00:08:03.280 --> 00:08:06.550 And you can always make the choice not to use a tag. 00:08:06.550 --> 00:08:11.110 So you saw some of that in the review article, right? 00:08:11.110 --> 00:08:13.390 And I guess one other thing, too, there's 00:08:13.390 --> 00:08:16.392 this idea of using the affinity tag in the affinity column, 00:08:16.392 --> 00:08:18.850 which we'll talk about more in the context of the ribosome, 00:08:18.850 --> 00:08:20.650 but is that always enough? 00:08:20.650 --> 00:08:24.250 So is the affinity column alone always enough 00:08:24.250 --> 00:08:27.520 to purify your protein of interest? 00:08:38.380 --> 00:08:40.600 So in lab class, that's where it will end, 00:08:40.600 --> 00:08:43.000 because that's an exercise made for lab class, 00:08:43.000 --> 00:08:45.880 and it's your first adventure into protein purification 00:08:45.880 --> 00:08:47.620 for most people, right? 00:08:47.620 --> 00:08:49.900 Oftentimes, it's not enough. 00:08:49.900 --> 00:08:53.590 That you do enrich what you've purified with what you want, 00:08:53.590 --> 00:08:55.000 but often there's contaminants. 00:08:55.000 --> 00:08:57.460 So you actually do move forward with doing some other type 00:08:57.460 --> 00:08:58.360 of purification. 00:08:58.360 --> 00:09:02.470 Like Rebecca mentioned, ion exchange or size exclusion, 00:09:02.470 --> 00:09:06.190 those are things you can use after the affinity purification 00:09:06.190 --> 00:09:08.830 there as needed, right? 00:09:08.830 --> 00:09:13.144 So contaminations are something to look out for here. 00:09:13.144 --> 00:09:14.810 JOANNE STUBBE: What other types of steps 00:09:14.810 --> 00:09:17.218 do you use for purification besides columns 00:09:17.218 --> 00:09:19.960 and [INAUDIBLE]. 00:09:19.960 --> 00:09:22.530 Because people have forgotten all of this. 00:09:22.530 --> 00:09:26.081 Everybody uses the tag and that's the end of it, 00:09:26.081 --> 00:09:28.740 and it can be the kiss of death. 00:09:28.740 --> 00:09:30.640 What other kind of fractionation steps? 00:09:30.640 --> 00:09:32.780 Do you do any other fractionation steps? 00:09:32.780 --> 00:09:35.050 What is it, 535 or something? 00:09:37.606 --> 00:09:38.730 AUDIENCE: [INAUDIBLE] salt. 00:09:38.730 --> 00:09:39.640 JOANNE STUBBE: The what? 00:09:39.640 --> 00:09:41.056 AUDIENCE: The [INAUDIBLE] salt. So 00:09:41.056 --> 00:09:42.700 some proteins will precipitate. 00:09:42.700 --> 00:09:43.687 Some will not. 00:09:43.687 --> 00:09:44.520 JOANNE STUBBE: Yeah. 00:09:44.520 --> 00:09:46.310 So that's what you use to precipitate. 00:09:46.310 --> 00:09:48.920 So that's a mild method. 00:09:48.920 --> 00:09:49.960 It's fast. 00:09:49.960 --> 00:09:55.190 It gives you separation on a fair amount of proteins. 00:09:55.190 --> 00:09:57.655 Anybody know what you use? 00:09:57.655 --> 00:09:58.780 AUDIENCE: Ammonium sulfate. 00:09:58.780 --> 00:10:00.450 JOANNE STUBBE: Yeah, ammonium sulfate. 00:10:00.450 --> 00:10:03.330 And then what's the other thing that really is important? 00:10:03.330 --> 00:10:05.830 What is the other thing you want to remove from your protein 00:10:05.830 --> 00:10:09.822 when you're using this tag that oftentimes people miss 00:10:09.822 --> 00:10:10.975 in the literature? 00:10:10.975 --> 00:10:12.850 ELIZABETH NOLAN: Yeah, we were getting there. 00:10:12.850 --> 00:10:15.183 JOANNE STUBBE: There's another component inside the cell 00:10:15.183 --> 00:10:18.751 that you need to get rid of that you've been talking about. 00:10:18.751 --> 00:10:22.202 AUDIENCE: Where one component has His-tag on the cell, 00:10:22.202 --> 00:10:24.522 you remove it. 00:10:24.522 --> 00:10:26.146 JOANNE STUBBE: So you have the protein. 00:10:26.146 --> 00:10:28.118 What are the other components in the cell 00:10:28.118 --> 00:10:30.090 that you need to remove? 00:10:30.090 --> 00:10:32.062 You can go back and look at your cartoon 00:10:32.062 --> 00:10:34.604 of the inside of the cell. 00:10:34.604 --> 00:10:35.520 ELIZABETH NOLAN: Yeah. 00:10:35.520 --> 00:10:38.560 So we're getting a little ahead, but that's totally fine. 00:10:38.560 --> 00:10:43.140 So if you're going to lyse your cell, and then, 00:10:43.140 --> 00:10:45.540 imagine your protein's soluble, right? 00:10:45.540 --> 00:10:48.090 So you do a centrifugation to remove 00:10:48.090 --> 00:10:49.500 the insoluble components. 00:10:49.500 --> 00:10:51.690 So you have the membrane and all that debris. 00:10:51.690 --> 00:10:53.790 And then you take your soluble fraction, 00:10:53.790 --> 00:10:57.000 and you incubate that on your column, right? 00:10:57.000 --> 00:11:00.367 And then you elute, you wash, you elute your protein. 00:11:00.367 --> 00:11:01.950 What might come out with your protein? 00:11:04.590 --> 00:11:05.650 AUDIENCE: Nucleic acid. 00:11:05.650 --> 00:11:06.858 ELIZABETH NOLAN: Yeah, right. 00:11:06.858 --> 00:11:10.866 So how do you know if you have nucleic acid contaminating? 00:11:10.866 --> 00:11:12.442 AUDIENCE: The A260. 00:11:12.442 --> 00:11:13.400 ELIZABETH NOLAN: Right. 00:11:13.400 --> 00:11:16.810 So A260 will give you a readout of nucleic acids. 00:11:16.810 --> 00:11:19.549 A280 is what people typically look at for their protein 00:11:19.549 --> 00:11:21.590 concentration, but you should look at both so you 00:11:21.590 --> 00:11:23.249 know what's in your protein. 00:11:23.249 --> 00:11:24.290 You need to look at both. 00:11:24.290 --> 00:11:26.282 JOANNE STUBBE: Do that and adaptively 00:11:26.282 --> 00:11:27.740 repeat stuff out of the literature. 00:11:27.740 --> 00:11:30.270 Very frequently, you take an absorption spectrum, 00:11:30.270 --> 00:11:31.460 there's some nucleic acid. 00:11:31.460 --> 00:11:33.126 ELIZABETH NOLAN: You have contamination. 00:11:33.126 --> 00:11:34.370 So a lot of ribosome, DNA. 00:11:34.370 --> 00:11:36.740 JOANNE STUBBE: If you don't remember anything else, 00:11:36.740 --> 00:11:37.580 it's important. 00:11:37.580 --> 00:11:38.840 ELIZABETH NOLAN: Yeah. 00:11:38.840 --> 00:11:42.150 So sure, just something to think about. 00:11:42.150 --> 00:11:47.882 So Alex noted, it's easy to put on this His-tag. 00:11:47.882 --> 00:11:50.780 What is actually on this His-tagged protein? 00:11:50.780 --> 00:11:54.077 So is it just the six histidines are all the same? 00:11:54.077 --> 00:11:55.910 When you look in the literature, and someone 00:11:55.910 --> 00:12:02.040 says they put a His-tag on the N terminus of their protein, 00:12:02.040 --> 00:12:03.220 how do you think about that? 00:12:10.684 --> 00:12:16.900 So we have some His6-tag, and then 00:12:16.900 --> 00:12:24.050 that tag, say, is attached to the protein here. 00:12:24.050 --> 00:12:25.225 What's going on in between? 00:12:28.480 --> 00:12:31.350 AUDIENCE: Maybe a flexible linker of some kind. 00:12:31.350 --> 00:12:32.350 ELIZABETH NOLAN: Pardon? 00:12:32.350 --> 00:12:33.280 AUDIENCE: It would be like a-- 00:12:33.280 --> 00:12:35.140 I don't know-- like a flexible linker of some kind. 00:12:35.140 --> 00:12:35.390 ELIZABETH NOLAN: Yeah. 00:12:35.390 --> 00:12:37.340 So maybe some kind of flexible linker. 00:12:37.340 --> 00:12:39.680 And what might dictate this linker? 00:12:47.616 --> 00:12:49.600 AUDIENCE: If you wanted to remove the His-tag 00:12:49.600 --> 00:12:53.452 after you would want to be able to hydrolyze it or something. 00:12:53.452 --> 00:12:54.910 ELIZABETH NOLAN: Yeah, so maybe you 00:12:54.910 --> 00:12:57.220 want to remove your tag down the road, right? 00:12:57.220 --> 00:12:59.770 So a protease is often employed. 00:12:59.770 --> 00:13:04.750 So maybe there's a linker, maybe there's a protease cleavage 00:13:04.750 --> 00:13:05.250 site. 00:13:09.980 --> 00:13:12.321 OK. 00:13:12.321 --> 00:13:12.820 OK. 00:13:12.820 --> 00:13:15.840 And we're not going to talk about cloning really 00:13:15.840 --> 00:13:20.650 in this class, but just thinking back a step, 00:13:20.650 --> 00:13:24.970 you need some plasmid DNA to ultimately get here 00:13:24.970 --> 00:13:29.240 that has your gene, right? 00:13:29.240 --> 00:13:32.260 And so you'll insert the gene into the plasmid, 00:13:32.260 --> 00:13:34.390 and many commercial plasmids have something 00:13:34.390 --> 00:13:36.260 called multiple cloning site. 00:13:36.260 --> 00:13:39.640 And, for instance, if you want a His-tag or a GST tag, 00:13:39.640 --> 00:13:41.650 you'll use some different plasmid 00:13:41.650 --> 00:13:43.720 that has that encoded, right? 00:13:43.720 --> 00:13:47.350 And then you make a decision about how you put your gene in. 00:13:47.350 --> 00:13:49.390 And these multiple cloning sites have 00:13:49.390 --> 00:13:51.430 multiple sites for restriction enzymes 00:13:51.430 --> 00:13:54.250 where you can put the gene in, which means, 00:13:54.250 --> 00:13:58.710 even if the same plasmid is used for 10 different proteins, 00:13:58.710 --> 00:14:02.200 what's happening here can vary a lot 00:14:02.200 --> 00:14:05.380 even if you have one protein and put it in different sites. 00:14:05.380 --> 00:14:09.880 So maybe you have a short linker here because you 00:14:09.880 --> 00:14:14.301 used a site like NDE1 or SPE1, I'm just making those up, 00:14:14.301 --> 00:14:14.800 right? 00:14:14.800 --> 00:14:17.350 Or maybe you have a longer region here 00:14:17.350 --> 00:14:19.110 between the tag and the protein. 00:14:19.110 --> 00:14:21.700 And so it's very important to go look back 00:14:21.700 --> 00:14:24.580 at the map of the plasmid that was used and ask 00:14:24.580 --> 00:14:27.550 where was the gene put in and what does that mean? 00:14:27.550 --> 00:14:30.730 So is this His-tag a 2 kilodalton perturbation. 00:14:30.730 --> 00:14:33.940 Is it a 5 kilodalton perterbation to your protein? 00:14:33.940 --> 00:14:39.520 And some of these plasmids have multiple types of tags, right? 00:14:39.520 --> 00:14:42.070 So it might have a GST tag and a His-tag, 00:14:42.070 --> 00:14:45.610 and depending how you put your gene in, you may have two 00:14:45.610 --> 00:14:47.620 or you may have only one, right? 00:14:47.620 --> 00:14:50.119 So I'm just pointing out there's a complexity here. 00:14:50.119 --> 00:14:51.910 So when someone just writes in their paper, 00:14:51.910 --> 00:14:53.710 oh, I His-tagged the protein, you 00:14:53.710 --> 00:14:56.740 need to think beyond just sticking six histamine residues 00:14:56.740 --> 00:14:58.480 on the N or C terminus there. 00:14:58.480 --> 00:15:00.358 Did you have a question? 00:15:00.358 --> 00:15:03.047 AUDIENCE: So does that mean that's like another step where 00:15:03.047 --> 00:15:06.226 they purified the specific plumbing site, 00:15:06.226 --> 00:15:07.693 one that they wanted? 00:15:07.693 --> 00:15:11.130 Or does that mean you kind of roll with the heterogeneous-- 00:15:11.130 --> 00:15:12.490 ELIZABETH NOLAN: No. 00:15:12.490 --> 00:15:16.090 So you put your gene in particular sites 00:15:16.090 --> 00:15:18.260 with this type of cloning, right? 00:15:18.260 --> 00:15:24.280 And so one thing practically, just say 00:15:24.280 --> 00:15:26.100 you want to express some new protein 00:15:26.100 --> 00:15:27.880 and you don't know much about this. 00:15:27.880 --> 00:15:31.120 You might choose to make several different constructs where 00:15:31.120 --> 00:15:33.760 you put the gene in different sites 00:15:33.760 --> 00:15:37.300 or maybe you use primers that allow you to add some linker 00:15:37.300 --> 00:15:39.910 regions because you don't know what will give you better 00:15:39.910 --> 00:15:42.420 solubility and better yield. 00:15:42.420 --> 00:15:45.730 And then ultimately, you pick one. 00:15:45.730 --> 00:15:52.760 So, for instance, like for me, with working with, say, 00:15:52.760 --> 00:15:56.650 a His-tag for a protein, there's plenty of plasmids 00:15:56.650 --> 00:16:00.040 available where you can pick N terminus or C terminus. 00:16:00.040 --> 00:16:02.770 So one plasmid to put the tag on the N terminus. 00:16:02.770 --> 00:16:05.530 A different plasmid to put the tag on the C terminus. 00:16:05.530 --> 00:16:08.890 I'll clone the gene into both and test overexpression 00:16:08.890 --> 00:16:11.020 with both and just see if one's better 00:16:11.020 --> 00:16:15.070 than the other in terms of yield, in terms of solubility 00:16:15.070 --> 00:16:16.180 there. 00:16:16.180 --> 00:16:17.800 And then you make a call. 00:16:17.800 --> 00:16:21.430 Maybe you purify both and see if there is an effect on behavior, 00:16:21.430 --> 00:16:25.670 like oligomerization or activity if it's an enzyme here. 00:16:25.670 --> 00:16:28.436 So if you get into protein purification, 00:16:28.436 --> 00:16:30.310 it's good to talk to people who have purified 00:16:30.310 --> 00:16:33.320 many different proteins because the strategy is 00:16:33.320 --> 00:16:35.300 a little different for each protein. 00:16:35.300 --> 00:16:37.270 And then you just get more exposure 00:16:37.270 --> 00:16:42.810 to all of the possibilities and troubleshooting there. 00:16:42.810 --> 00:16:49.000 So coming to the paper, what was the big motivation 00:16:49.000 --> 00:16:53.920 for developing this method to have an affinity tag attached 00:16:53.920 --> 00:16:57.140 to the ribosome? 00:16:57.140 --> 00:16:57.640 Right? 00:16:57.640 --> 00:17:01.030 So Youngman and Green went to quite a bit of effort 00:17:01.030 --> 00:17:02.320 to devise this new system. 00:17:04.877 --> 00:17:05.960 What was their motivation? 00:17:10.055 --> 00:17:19.420 And what really was the big issue 00:17:19.420 --> 00:17:22.882 they were seeking to overcome? 00:17:22.882 --> 00:17:26.340 AUDIENCE: They wanted to get ribosomes with mutations on it. 00:17:26.340 --> 00:17:30.084 So they want to synthesize bacteria ribosomes with it 00:17:30.084 --> 00:17:30.964 and purify it. 00:17:30.964 --> 00:17:31.880 ELIZABETH NOLAN: Yeah. 00:17:31.880 --> 00:17:33.740 So they want a mutant ribosome. 00:17:38.270 --> 00:17:40.860 And they want to make this mutant ribosome 00:17:40.860 --> 00:17:47.810 in vivo and then purify. 00:17:50.420 --> 00:17:54.750 So what's the complication with making the mutant ribosome 00:17:54.750 --> 00:17:59.948 in vivo that they seek to overcome here? 00:17:59.948 --> 00:18:03.260 AUDIENCE: You have the wild-type ribosomes in there also. 00:18:03.260 --> 00:18:04.729 ELIZABETH NOLAN: So well, right. 00:18:04.729 --> 00:18:06.020 That was their decision, right? 00:18:06.020 --> 00:18:09.560 They want to express this mutant ribosome in the background 00:18:09.560 --> 00:18:11.000 of the wild-type ribosome. 00:18:11.000 --> 00:18:14.048 So why do they want to do that? 00:18:14.048 --> 00:18:15.952 AUDIENCE: Because it could be lethal. 00:18:15.952 --> 00:18:20.712 The mutant ribosome, it would be a toxic mutant. 00:18:20.712 --> 00:18:22.910 ELIZABETH NOLAN: Yeah, toxic mutant. 00:18:22.910 --> 00:18:24.806 What do you mean by "toxic mutant?" 00:18:24.806 --> 00:18:28.915 AUDIENCE: If you create a mutant ribosome, that 00:18:28.915 --> 00:18:32.806 was the only way for the cell to express them, they'd be toxic 00:18:32.806 --> 00:18:35.234 and they wouldn't be able to go through translation. 00:18:35.234 --> 00:18:36.150 ELIZABETH NOLAN: Yeah. 00:18:36.150 --> 00:18:39.240 So maybe the mutant ribosome doesn't work very well, 00:18:39.240 --> 00:18:43.260 and that ends up being lethal to the cell there. 00:18:43.260 --> 00:18:47.250 So can we imagine why that might be 00:18:47.250 --> 00:18:49.770 an issue for the types of experiments 00:18:49.770 --> 00:18:51.040 we've seen in class? 00:18:51.040 --> 00:18:53.370 So if you're thinking about trying 00:18:53.370 --> 00:18:57.690 to understand the catalytic mechanism in that function, 00:18:57.690 --> 00:19:01.620 there's a likelihood the mutations may dramatically 00:19:01.620 --> 00:19:03.180 affect that activity, right? 00:19:03.180 --> 00:19:04.770 If you want to put a point mutation 00:19:04.770 --> 00:19:07.060 into the peptidyl transferase center, 00:19:07.060 --> 00:19:09.060 into the decoding center, that could 00:19:09.060 --> 00:19:11.760 be deleterious to your cell, but it 00:19:11.760 --> 00:19:14.700 could be very important for your mechanistic study. 00:19:14.700 --> 00:19:18.240 So they want to avoid this lethal phenotype. 00:19:29.250 --> 00:19:32.810 So what is the complication in terms 00:19:32.810 --> 00:19:39.560 of doing this in the presence of wild-type 00:19:39.560 --> 00:19:42.790 for some sort of measurement? 00:19:42.790 --> 00:19:44.381 AUDIENCE: It gets kind of muddy. 00:19:44.381 --> 00:19:45.880 ELIZABETH NOLAN: Gets kind of muddy. 00:19:45.880 --> 00:19:46.260 Yeah. 00:19:46.260 --> 00:19:47.093 What does that mean? 00:19:47.093 --> 00:19:50.583 AUDIENCE: You don't have a pure mutant in there, 00:19:50.583 --> 00:19:56.134 and they're not significantly different from the wild-type. 00:19:56.134 --> 00:19:57.050 ELIZABETH NOLAN: Yeah. 00:19:57.050 --> 00:19:59.180 So if you were going to do a standard ribosome 00:19:59.180 --> 00:20:00.350 purification-- 00:20:00.350 --> 00:20:02.690 because ribosomes have been purified for many years 00:20:02.690 --> 00:20:04.265 without an affinity tag-- 00:20:04.265 --> 00:20:06.140 you're going to have a mixture of your mutant 00:20:06.140 --> 00:20:07.730 and the wild-type. 00:20:07.730 --> 00:20:11.630 And so that has a strong likelihood 00:20:11.630 --> 00:20:14.300 of being a problem for your analysis, right? 00:20:14.300 --> 00:20:16.100 So they gave an example in this paper 00:20:16.100 --> 00:20:17.540 where they actually made a mixture 00:20:17.540 --> 00:20:20.390 and did some analyses, where you could separate 00:20:20.390 --> 00:20:22.290 the wild-type from mutant activity 00:20:22.290 --> 00:20:24.840 but that's not necessarily the case. 00:20:24.840 --> 00:20:28.350 And so, as pointed out, they're both very large. 00:20:28.350 --> 00:20:30.110 They're very similar, right? 00:20:30.110 --> 00:20:33.500 There's no good way to separate a ribosome 00:20:33.500 --> 00:20:35.060 with a single-point mutation, say, 00:20:35.060 --> 00:20:38.100 in the peptidyl transferase center from wild-type. 00:20:38.100 --> 00:20:45.110 So let's just imagine you have a mixture that's predominantly 00:20:45.110 --> 00:20:48.050 your mutant ribosome but you have some background 00:20:48.050 --> 00:20:49.820 contamination of wild-type. 00:20:49.820 --> 00:20:50.600 Is that an issue? 00:20:58.536 --> 00:21:05.580 Say you want to measure rates of peptide bond formation. 00:21:05.580 --> 00:21:07.080 AUDIENCE: It's going to be an issue. 00:21:07.080 --> 00:21:07.996 ELIZABETH NOLAN: Yeah. 00:21:07.996 --> 00:21:09.941 So why is it probably going to be an issue? 00:21:09.941 --> 00:21:11.624 AUDIENCE: Because your mutant might 00:21:11.624 --> 00:21:15.232 be a lethal function because of your background 00:21:15.232 --> 00:21:20.094 and resume the function and [INAUDIBLE] mutant [INAUDIBLE].. 00:21:20.094 --> 00:21:21.010 ELIZABETH NOLAN: Yeah. 00:21:21.010 --> 00:21:23.010 So imagine you have a mutant ribosome that 00:21:23.010 --> 00:21:27.660 has very low activity or none, and you 00:21:27.660 --> 00:21:30.030 have some small amount of contaminating wild-type that 00:21:30.030 --> 00:21:32.610 has wild-type activity, right? 00:21:32.610 --> 00:21:34.200 How do you know-- 00:21:34.200 --> 00:21:36.420 I mean you might misinterpret your data, 00:21:36.420 --> 00:21:39.330 and what you're seeing is the wild-type and not the mutant. 00:21:39.330 --> 00:21:41.880 And this issue is much more broad than the ribosome. 00:21:41.880 --> 00:21:45.240 So one of my favorites is the contaminating ATPase. 00:21:45.240 --> 00:21:48.810 And maybe you have an enzyme that hydrolyzes ATP, 00:21:48.810 --> 00:21:50.640 but maybe you have a small contamination 00:21:50.640 --> 00:21:54.870 of an enzyme that does a much better job at hydrolyzing ATP 00:21:54.870 --> 00:21:56.670 that's in your reaction, right? 00:21:56.670 --> 00:21:59.400 So what are you seeing there? 00:21:59.400 --> 00:22:01.140 So that's something to keep in mind 00:22:01.140 --> 00:22:04.920 in terms of potential contaminations, right? 00:22:04.920 --> 00:22:06.900 And so they go through some justification 00:22:06.900 --> 00:22:09.270 about why they need to do this method based 00:22:09.270 --> 00:22:13.020 on available methods, and all of those available methods 00:22:13.020 --> 00:22:14.410 have strengths and weaknesses. 00:22:14.410 --> 00:22:16.080 So they talked about using systems 00:22:16.080 --> 00:22:18.260 without the wild-type ribosome. 00:22:18.260 --> 00:22:20.250 They mentioned in vitro translation, 00:22:20.250 --> 00:22:25.950 and I'll just say, in passing, those in vitro systems 00:22:25.950 --> 00:22:29.861 have improved a lot since the time of this paper. 00:22:29.861 --> 00:22:30.360 OK. 00:22:30.360 --> 00:22:33.520 So in terms of their strategy. 00:22:33.520 --> 00:22:40.010 Let's comment on the various aspects of this strategy. 00:22:40.010 --> 00:22:40.510 All right. 00:22:40.510 --> 00:22:43.650 So effectively, they want a way to express 00:22:43.650 --> 00:22:48.150 the mutant ribosome in vivo in the background of wild-type. 00:22:48.150 --> 00:22:50.730 They want a way to separate that ribosome. 00:22:50.730 --> 00:22:53.951 And they want to come up with ribosomes that are active. 00:22:53.951 --> 00:22:54.450 OK. 00:22:54.450 --> 00:22:58.800 So this cartoon basically summarizes their solution 00:22:58.800 --> 00:23:01.920 to this problem, and we should work 00:23:01.920 --> 00:23:06.010 through the various components. 00:23:06.010 --> 00:23:09.450 So the first thing is they attached a tag 00:23:09.450 --> 00:23:12.570 to either the 23S or the 16S. 00:23:12.570 --> 00:23:15.457 And we'll focus today's discussion on the 23S 00:23:15.457 --> 00:23:17.540 because that's what they did more characterization 00:23:17.540 --> 00:23:18.990 on in the paper. 00:23:18.990 --> 00:23:23.000 So how did they decide on the tag and where to place the tag? 00:23:27.930 --> 00:23:29.902 AUDIENCE: You don't want the tag somewhere 00:23:29.902 --> 00:23:32.367 that's going to interfere with function. 00:23:32.367 --> 00:23:34.832 And you don't want the tag itself to be reactive 00:23:34.832 --> 00:23:36.311 where it will interfere. 00:23:36.311 --> 00:23:41.241 So you need to be out of the way and kind of passive 00:23:41.241 --> 00:23:43.127 so it's not interfering with function. 00:23:43.127 --> 00:23:44.960 ELIZABETH NOLAN: So that's one point, right? 00:23:44.960 --> 00:23:48.660 We don't want this tag to interfere with function. 00:23:48.660 --> 00:23:50.970 So that's one aspect. 00:23:50.970 --> 00:23:52.752 What's another aspect? 00:23:52.752 --> 00:23:54.460 AUDIENCE: You still have to be accessible 00:23:54.460 --> 00:23:55.960 ELIZABETH NOLAN: Yeah, the tag needs 00:23:55.960 --> 00:23:57.510 to be accessible because something's 00:23:57.510 --> 00:24:00.350 going to have to bind this tag, right? 00:24:00.350 --> 00:24:04.220 So on the basis of those two criteria, 00:24:04.220 --> 00:24:07.350 we can imagine wanting this tag somewhere on the surface, 00:24:07.350 --> 00:24:07.850 right? 00:24:07.850 --> 00:24:11.030 We don't want it where the 30S and 50S interact. 00:24:11.030 --> 00:24:13.820 We don't want it in a position that's critical. 00:24:13.820 --> 00:24:16.330 So like maybe if it ended up near where 00:24:16.330 --> 00:24:19.350 EFTU first binds or EFG, that would be bad. 00:24:19.350 --> 00:24:23.630 So accessible and in a place where it won't interfere. 00:24:23.630 --> 00:24:26.510 Beyond that, the ribosome's huge. 00:24:26.510 --> 00:24:29.360 So how does one pick where to put this tag? 00:24:29.360 --> 00:24:30.650 What did the researchers do? 00:24:37.134 --> 00:24:38.550 I'll tell you what they didn't do. 00:24:38.550 --> 00:24:40.240 They didn't reinvent the wheel. 00:24:40.240 --> 00:24:43.560 So what did they do? 00:24:43.560 --> 00:24:44.544 AUDIENCE: Lit review. 00:24:44.544 --> 00:24:45.460 ELIZABETH NOLAN: Yeah. 00:24:45.460 --> 00:24:46.626 They went to the literature. 00:24:46.626 --> 00:24:48.606 And what did they find in the literature? 00:24:52.378 --> 00:24:54.794 AUDIENCE: Someone had installed a tRNA before or something 00:24:54.794 --> 00:24:57.142 and it didn't interfere with the ribosome function. 00:24:57.142 --> 00:24:58.100 ELIZABETH NOLAN: Right. 00:24:58.100 --> 00:25:00.970 So they took observations that were 00:25:00.970 --> 00:25:04.990 made from an independent group for an independent project 00:25:04.990 --> 00:25:08.150 but the observations that were useful to them in their design. 00:25:08.150 --> 00:25:12.460 So for some reason, this lab stuck a tRNA onto the ribosome 00:25:12.460 --> 00:25:16.060 and saw the ribosome was still active and functioning well. 00:25:16.060 --> 00:25:18.280 So the decision was, why don't we 00:25:18.280 --> 00:25:21.890 use that to place the tag here? 00:25:21.890 --> 00:25:25.180 So what about their choice of tag? 00:25:25.180 --> 00:25:25.945 What did they use? 00:25:30.457 --> 00:25:32.380 A big tag or a little tag? 00:25:37.528 --> 00:25:40.590 Any sense of that compared to the size of, say, the 50S? 00:25:46.840 --> 00:25:48.190 Take a look at figure one. 00:26:07.152 --> 00:26:09.647 AUDIENCE: A small. 00:26:09.647 --> 00:26:11.870 ELIZABETH NOLAN: Yeah, right. 00:26:11.870 --> 00:26:16.280 So I'd say very small compared to the size 00:26:16.280 --> 00:26:17.750 of the ribosome, right? 00:26:17.750 --> 00:26:20.360 So they decided to take advantage of interaction 00:26:20.360 --> 00:26:27.050 between this MS2 coat protein and the MS2 RNA recognition 00:26:27.050 --> 00:26:28.610 sequence, right? 00:26:28.610 --> 00:26:34.160 So that's one interaction involved here, 00:26:34.160 --> 00:26:35.820 so ligand receptor interaction. 00:26:35.820 --> 00:26:39.770 So here, this is the depiction from the paper showing 00:26:39.770 --> 00:26:48.075 where they incorporated this MS2 stem loop into the 23S rRNA, 00:26:48.075 --> 00:26:49.100 here. 00:26:49.100 --> 00:26:57.075 And so what does that tag need to bind, going back 00:26:57.075 --> 00:26:57.700 to the cartoon? 00:27:03.970 --> 00:27:06.290 And what is different about this strategy 00:27:06.290 --> 00:27:09.680 from, say, what you've done with nickel and TA chromatography 00:27:09.680 --> 00:27:12.050 and His6-tags? 00:27:12.050 --> 00:27:13.400 AUDIENCE: It has three things. 00:27:13.400 --> 00:27:14.970 ELIZABETH NOLAN: Three things, yeah. 00:27:14.970 --> 00:27:15.470 Right? 00:27:15.470 --> 00:27:20.300 So we have three components, two different interactions, 00:27:20.300 --> 00:27:24.170 say, between a ligand and its binding partner, right? 00:27:24.170 --> 00:27:30.860 So here, we have the mutant RNA of the ribosome where 00:27:30.860 --> 00:27:34.610 there's this MS2 stem loop, OK? 00:27:34.610 --> 00:27:39.680 That MS2 stem loop binds to the MS2 coat protein, right? 00:27:39.680 --> 00:27:42.560 And then there needs to be some way to pull this out. 00:27:42.560 --> 00:27:44.660 And what they chose to do here was 00:27:44.660 --> 00:27:46.940 take advantage of a second interaction and one 00:27:46.940 --> 00:27:50.330 that's commonly used in chemistry and biology, which 00:27:50.330 --> 00:27:53.780 is looking at an interaction between a protein called 00:27:53.780 --> 00:28:00.380 glutathione S-transferase and glutathione, here, right? 00:28:00.380 --> 00:28:03.330 So effectively, they have a solid support or a resin, 00:28:03.330 --> 00:28:05.690 so like the nickel NTA column, but in this case, 00:28:05.690 --> 00:28:08.090 it's modified with GSH. 00:28:08.090 --> 00:28:11.420 They have to prepare this fusion protein that 00:28:11.420 --> 00:28:15.350 is a fusion of GST and MS2, and then they 00:28:15.350 --> 00:28:17.060 have the ribosome with the tag. 00:28:20.380 --> 00:28:22.960 So why might they have done this with three components 00:28:22.960 --> 00:28:23.910 rather than two? 00:28:28.812 --> 00:28:35.660 AUDIENCE: Since GST/GSH, these are pretty standard affinity 00:28:35.660 --> 00:28:39.395 tags, it was probably easier to acquire GSH then 00:28:39.395 --> 00:28:40.624 to try to get MS2. 00:28:40.624 --> 00:28:41.540 ELIZABETH NOLAN: Yeah. 00:28:41.540 --> 00:28:46.420 That's a practical analysis there. 00:28:46.420 --> 00:28:51.150 So could they have made a resin with MS2? 00:28:51.150 --> 00:28:54.240 Maybe, right? 00:28:54.240 --> 00:29:04.190 So how did they go about doing this affinity purification? 00:29:04.190 --> 00:29:06.650 What were the steps and why? 00:29:12.968 --> 00:29:19.660 So imagine they've done the molecular biology required 00:29:19.660 --> 00:29:22.000 to express this tagged ribosome. 00:29:22.000 --> 00:29:26.020 They expressed the tagged ribosome in E. coli. 00:29:26.020 --> 00:29:26.530 Then what? 00:29:26.530 --> 00:29:30.750 How are they going to get this tagged ribosome out? 00:29:36.510 --> 00:29:39.870 AUDIENCE: They first purify the crude ribosomes [INAUDIBLE] 00:29:39.870 --> 00:29:42.758 including the [INAUDIBLE] and normal ones. 00:29:42.758 --> 00:29:47.520 And then they load these crude samples through the column. 00:29:47.520 --> 00:29:48.620 ELIZABETH NOLAN: OK. 00:29:48.620 --> 00:29:51.470 So the crude sample went through the column, right? 00:29:51.470 --> 00:29:54.050 What happened before that? 00:29:54.050 --> 00:29:57.440 So can you just put the crude ribosome through the column, 00:29:57.440 --> 00:30:01.309 if your column is the resin with GSH? 00:30:01.309 --> 00:30:03.600 AUDIENCE: You have to preload it with a fusion protein. 00:30:03.600 --> 00:30:04.670 ELIZABETH NOLAN: Yes. 00:30:04.670 --> 00:30:07.334 So what did they preload with the fusion protein? 00:30:10.652 --> 00:30:11.600 AUDIENCE: The column. 00:30:11.600 --> 00:30:12.475 ELIZABETH NOLAN: Yes. 00:30:12.475 --> 00:30:14.020 That's what they did, right? 00:30:14.020 --> 00:30:17.320 We can imagine just looking at this without further details. 00:30:17.320 --> 00:30:21.160 There's two possibilities with three components. 00:30:21.160 --> 00:30:25.120 So they could have, as stated and as what they finally did, 00:30:25.120 --> 00:30:28.180 they could add this fusion protein to the column, right? 00:30:28.180 --> 00:30:31.150 So the fusion protein binds GSH. 00:30:31.150 --> 00:30:36.640 And then you take your crude lysate, crude material 00:30:36.640 --> 00:30:40.690 from the E. coli and run that through the column 00:30:40.690 --> 00:30:42.880 to trap the ribosomes. 00:30:42.880 --> 00:30:44.350 What's the other possibility? 00:30:47.312 --> 00:30:49.020 They could have taken this fusion protein 00:30:49.020 --> 00:30:51.660 and put it into the crude mixture, 00:30:51.660 --> 00:30:52.920 and they talked about that. 00:30:52.920 --> 00:30:55.860 So what was one of the complications with this fusion 00:30:55.860 --> 00:30:59.055 protein that led them to incubate it with the column? 00:31:04.050 --> 00:31:06.370 Was this fusion protein well-behaved? 00:31:26.010 --> 00:31:29.182 AUDIENCE: It forms insoluble aggregate. 00:31:29.182 --> 00:31:30.390 ELIZABETH NOLAN: Yeah, right? 00:31:30.390 --> 00:31:32.480 It gave them some headaches. 00:31:32.480 --> 00:31:36.090 It aggregated. 00:31:36.090 --> 00:31:38.105 Is that something that can commonly happen? 00:31:46.240 --> 00:31:50.320 So they likely tried both ways, and they observed this problem 00:31:50.320 --> 00:31:54.790 with the fusion protein having aggregation, right? 00:31:54.790 --> 00:31:57.670 And they avoided that as being a complication 00:31:57.670 --> 00:32:00.430 to the purification by incubating the column 00:32:00.430 --> 00:32:03.670 with that fusion protein first. 00:32:03.670 --> 00:32:09.100 So after they take their crude sample 00:32:09.100 --> 00:32:12.400 and have that bind to the resin in the column, 00:32:12.400 --> 00:32:18.200 how did they get the ribosomes off the column? 00:32:18.200 --> 00:32:20.180 So what are the possibilities? 00:32:20.180 --> 00:32:22.420 And what did they end up doing and why? 00:32:28.144 --> 00:32:32.190 AUDIENCE: So you could just use an excessive of free MS2 00:32:32.190 --> 00:32:34.630 ligands to cause the ribosomes to disassociate 00:32:34.630 --> 00:32:35.572 from the column. 00:32:35.572 --> 00:32:37.927 Or you could use an excess of the GST 00:32:37.927 --> 00:32:40.784 to cause that complex to dissociate from the column. 00:32:40.784 --> 00:32:41.700 ELIZABETH NOLAN: Yeah. 00:32:41.700 --> 00:32:46.590 So thinking about the latter possibility-- 00:32:46.590 --> 00:32:48.660 so what Rebecca has done is identify 00:32:48.660 --> 00:32:53.340 the two different ligand receptor interactions, right? 00:32:53.340 --> 00:32:57.450 We could disrupt this one between MS2 fusion coat 00:32:57.450 --> 00:33:01.710 protein and the ribosome or between GSH and GST. 00:33:01.710 --> 00:33:06.690 So in terms of this one here, does it make more sense 00:33:06.690 --> 00:33:12.510 to elute with excess protein or excess glutathione, which 00:33:12.510 --> 00:33:14.100 is effectively a tripeptide? 00:33:17.514 --> 00:33:18.430 AUDIENCE: Glutathione. 00:33:18.430 --> 00:33:19.638 ELIZABETH NOLAN: Yeah, right? 00:33:19.638 --> 00:33:24.790 So just that much easier to come by a lot of glutathione 00:33:24.790 --> 00:33:26.510 than a lot of GSH-- 00:33:26.510 --> 00:33:28.150 or sorry, GST. 00:33:28.150 --> 00:33:29.750 So which one did they choose? 00:33:34.115 --> 00:33:36.690 How did they elute the ribosome off the column? 00:33:42.460 --> 00:33:45.700 How long did you each spend on the paper before coming here? 00:33:48.538 --> 00:33:49.960 AUDIENCE: GSH. 00:33:49.960 --> 00:33:52.120 ELIZABETH NOLAN: Yeah, they used GSH. 00:33:52.120 --> 00:33:55.090 So they eluted with excess GSH. 00:33:55.090 --> 00:34:00.100 So what does that mean in terms of the purified ribosome? 00:34:00.100 --> 00:34:02.110 Is it just the ribosome with the stem loop? 00:34:02.110 --> 00:34:03.060 AUDIENCE: They still have the fusion protein. 00:34:03.060 --> 00:34:04.018 ELIZABETH NOLAN: Right. 00:34:04.018 --> 00:34:06.460 We still have the fusion protein on. 00:34:06.460 --> 00:34:10.540 So if you were making this decision at the bench, 00:34:10.540 --> 00:34:14.320 you have two different interactions to consider, 00:34:14.320 --> 00:34:17.266 what would you choose and why? 00:34:17.266 --> 00:34:19.030 Why and I guess, really, why did they 00:34:19.030 --> 00:34:22.530 choose to disrupt the interaction between GSH 00:34:22.530 --> 00:34:23.030 and GST? 00:34:29.740 --> 00:34:36.600 AUDIENCE: Because it's difficult to have enough MS2 than 00:34:36.600 --> 00:34:38.042 to purify with glutathione. 00:34:38.042 --> 00:34:39.250 ELIZABETH NOLAN: Here, right? 00:34:39.250 --> 00:34:44.080 Because if you were going to, say, elute with excess of MS2 00:34:44.080 --> 00:34:47.620 stem loop, where would that come from? 00:34:47.620 --> 00:34:50.100 Or excess MS2 protein, right? 00:34:50.100 --> 00:34:51.935 AUDIENCE: Also, it's actually MS2. 00:34:51.935 --> 00:34:52.810 ELIZABETH NOLAN: Yes. 00:34:52.810 --> 00:34:55.630 It's not very practical here, right? 00:34:55.630 --> 00:34:57.760 At the end of the day, it would be best 00:34:57.760 --> 00:35:00.880 to have the ribosome without this fusion protein attached, 00:35:00.880 --> 00:35:04.030 but disrupting this interaction isn't very practical. 00:35:04.030 --> 00:35:06.610 So it would be quite expensive either way 00:35:06.610 --> 00:35:08.787 if you were making a lot of some sort of stem loop. 00:35:08.787 --> 00:35:10.870 And then how would you even know you have the stem 00:35:10.870 --> 00:35:14.750 loop or the protein here? 00:35:14.750 --> 00:35:15.850 OK. 00:35:15.850 --> 00:35:18.790 So I'm just curious, for those of you 00:35:18.790 --> 00:35:21.670 who have done like nickel NTA chromatography, 00:35:21.670 --> 00:35:26.320 do you have a sense of the affinity of the His-tag protein 00:35:26.320 --> 00:35:29.030 for the resin? 00:35:29.030 --> 00:35:32.425 So what happens as this tied protein goes down the column? 00:35:40.165 --> 00:35:40.670 All right. 00:35:40.670 --> 00:35:58.286 So you have some column with your resin plus His6 protein. 00:36:13.036 --> 00:36:17.482 So was it a strong or weak interaction? 00:36:17.482 --> 00:36:18.190 AUDIENCE: Strong. 00:36:18.190 --> 00:36:19.110 AUDIENCE: Strong. 00:36:19.110 --> 00:36:21.526 ELIZABETH NOLAN: How would you define strong? 00:36:21.526 --> 00:36:23.896 Or why do you say it's strong? 00:36:23.896 --> 00:36:26.429 AUDIENCE: The chelation. 00:36:26.429 --> 00:36:28.720 ELIZABETH NOLAN: Well, you're forming a complex, right? 00:36:28.720 --> 00:36:32.330 You're forming-- the His-tag is binding the nickel NTA. 00:36:32.330 --> 00:36:36.470 AUDIENCE: The Kd is probably [INAUDIBLE].. 00:36:36.470 --> 00:36:37.866 ELIZABETH NOLAN: Is it? 00:36:37.866 --> 00:36:39.285 AUDIENCE: I don't know. 00:36:39.285 --> 00:36:41.028 AUDIENCE: It is a dynamic process 00:36:41.028 --> 00:36:43.069 where they're releasing and binding and releasing 00:36:43.069 --> 00:36:44.252 and binding again. 00:36:46.850 --> 00:36:47.900 ELIZABETH NOLAN: Yes. 00:36:47.900 --> 00:36:49.920 So there's an equilibrium, right? 00:36:49.920 --> 00:36:53.080 And we talk about binding to the column 00:36:53.080 --> 00:36:56.720 but how tightly is this tagged protein binding? 00:36:56.720 --> 00:36:58.850 And is it just binding there and getting stuck? 00:36:58.850 --> 00:37:02.360 I mean, it needs to stay in your column, right? 00:37:02.360 --> 00:37:05.190 In this case, it's not very strong. 00:37:05.190 --> 00:37:07.580 So if you look at reported Kd's for, say, 00:37:07.580 --> 00:37:09.890 His-tags to nickel NTA, they're on the order 00:37:09.890 --> 00:37:12.470 of one to 10 micromolar. 00:37:12.470 --> 00:37:16.175 So orders of magnitude lower affinity 00:37:16.175 --> 00:37:17.900 than what you just suggested. 00:37:17.900 --> 00:37:21.970 So why does the column work? 00:37:21.970 --> 00:37:24.419 AUDIENCE: Because everything else binds worse than that. 00:37:24.419 --> 00:37:25.960 ELIZABETH NOLAN: Well, you hope that. 00:37:25.960 --> 00:37:26.459 You hope. 00:37:26.459 --> 00:37:28.312 I mean, sure. 00:37:28.312 --> 00:37:30.520 I mean, if you know that histamine-- a protein that's 00:37:30.520 --> 00:37:34.720 histamine-rich it's going to stick, but why does it work? 00:37:37.750 --> 00:37:41.860 So is it surprising that a micromolar affinity 00:37:41.860 --> 00:37:46.300 can allow this to be trapped on the column? 00:37:46.300 --> 00:37:49.730 AUDIENCE: Is it because you have six histamines tied down 00:37:49.730 --> 00:37:56.080 [INAUDIBLE] 00:37:56.080 --> 00:37:57.080 ELIZABETH NOLAN: Pardon? 00:38:00.510 --> 00:38:01.030 OK. 00:38:01.030 --> 00:38:04.580 It's not the amount of histamines. 00:38:04.580 --> 00:38:06.687 What is in your column? 00:38:06.687 --> 00:38:08.520 AUDIENCE: You've got a lot of binding sites. 00:38:08.520 --> 00:38:08.850 ELIZABETH NOLAN: Yeah. 00:38:08.850 --> 00:38:09.430 Right. 00:38:09.430 --> 00:38:13.220 There's a lot of binding sites in the column. 00:38:13.220 --> 00:38:15.410 So you're going to have, as Rebecca said, dynamic. 00:38:15.410 --> 00:38:17.212 This is coming on and off the column, 00:38:17.212 --> 00:38:18.670 but there's a lot of binding sites. 00:38:18.670 --> 00:38:21.520 So if it comes off, it can go back on there. 00:38:25.822 --> 00:38:29.215 So what about the GST and GSH? 00:38:35.450 --> 00:38:37.925 AUDIENCE: I know it's one of the strongest attractions. 00:38:37.925 --> 00:38:38.841 ELIZABETH NOLAN: Yeah. 00:38:38.841 --> 00:38:40.860 This is much stronger than nickel NTA 00:38:40.860 --> 00:38:42.600 and the His-tag protein. 00:38:42.600 --> 00:38:46.766 So orders of magnitude higher affinity here for that. 00:38:46.766 --> 00:38:47.536 OK. 00:38:47.536 --> 00:38:49.410 But it's something to think about when you're 00:38:49.410 --> 00:38:54.384 choosing an affinity purification method there 00:38:54.384 --> 00:38:55.800 and to think about what's actually 00:38:55.800 --> 00:39:02.330 happening on this column and dynamic process. 00:39:02.330 --> 00:39:09.780 So jumping ahead a little bit, they did their experiments 00:39:09.780 --> 00:39:13.622 first just tagging the wild-type ribosome, OK? 00:39:13.622 --> 00:39:15.330 And that's very important because they're 00:39:15.330 --> 00:39:17.790 trying to make a new purification method, 00:39:17.790 --> 00:39:19.980 and the first thing that needs to be asked 00:39:19.980 --> 00:39:23.940 is does the wild-type ribosome plus the tag 00:39:23.940 --> 00:39:28.470 behave the same or differently from the wild-type ribosome 00:39:28.470 --> 00:39:30.330 without the tag, OK? 00:39:30.330 --> 00:39:32.370 And you want to know that because if the tag is 00:39:32.370 --> 00:39:34.702 causing a problem, maybe it's not a good design. 00:39:34.702 --> 00:39:36.660 And you don't want to go forward making mutants 00:39:36.660 --> 00:39:41.100 with that kind of modification, OK? 00:39:41.100 --> 00:39:49.500 So what do they need to do after doing this purification, right? 00:39:49.500 --> 00:39:53.910 One is just analyzing the purity of the material 00:39:53.910 --> 00:39:55.440 that they've come up with. 00:39:55.440 --> 00:39:57.390 And then the other things they did 00:39:57.390 --> 00:39:59.940 was look at the subunit integrity, right? 00:39:59.940 --> 00:40:03.000 And so something to keep in mind is that the ribosome 00:40:03.000 --> 00:40:04.050 has two subunits. 00:40:04.050 --> 00:40:07.110 It has many ribosomal proteins. 00:40:07.110 --> 00:40:10.770 Does putting the tag on only one subunit work well? 00:40:10.770 --> 00:40:14.490 And then, of course, they need to think about the activity. 00:40:14.490 --> 00:40:16.770 And so they presented a number of different assays 00:40:16.770 --> 00:40:21.060 in this paper ranging from looking at kinetics 00:40:21.060 --> 00:40:23.730 of peptide bond formation to looking 00:40:23.730 --> 00:40:29.100 at kinetic studies of release with one of the release factors 00:40:29.100 --> 00:40:30.280 here. 00:40:30.280 --> 00:40:34.590 So let's think about the purity analysis. 00:40:34.590 --> 00:40:38.310 And what we're going to focus on is there chromatogram, right? 00:40:38.310 --> 00:40:43.680 So as we know, they took their column of GSH. 00:40:43.680 --> 00:40:47.130 They first loaded that column with the GST/MS2 fusion 00:40:47.130 --> 00:40:52.080 protein, and then they added their crude sample 00:40:52.080 --> 00:40:54.580 and eluted with GSH, right? 00:40:54.580 --> 00:40:58.410 And so the data they present for the chromatogram for monitoring 00:40:58.410 --> 00:41:02.370 fractions of that column is shown here, OK? 00:41:02.370 --> 00:41:04.980 So what does this tell us? 00:41:04.980 --> 00:41:08.237 What do we see in this chromatogram? 00:41:13.704 --> 00:41:15.636 So what are they monitoring? 00:41:21.980 --> 00:41:25.270 So first you want to read your axes, right? 00:41:25.270 --> 00:41:27.602 So what are they monitoring. 00:41:27.602 --> 00:41:28.974 AUDIENCE: A260. 00:41:28.974 --> 00:41:30.640 ELIZABETH NOLAN: Yes, that's the y-axis. 00:41:30.640 --> 00:41:32.290 And why A260? 00:41:32.290 --> 00:41:34.654 Going back to 20 minutes ago. 00:41:34.654 --> 00:41:35.410 AUDIENCE: DNA. 00:41:35.410 --> 00:41:36.910 ELIZABETH NOLAN: Nucleotides, right? 00:41:36.910 --> 00:41:38.340 Do we want to monitor DNA here? 00:41:41.260 --> 00:41:42.730 It will work for DNA. 00:41:42.730 --> 00:41:43.614 AUDIENCE: Oh, RNA 00:41:43.614 --> 00:41:44.530 ELIZABETH NOLAN: Yeah. 00:41:44.530 --> 00:41:48.850 So we have the 23S, 16S rRNA. 00:41:48.850 --> 00:41:51.430 So we're looking at A260, which makes sense. 00:41:51.430 --> 00:41:54.600 We're trying to purify the ribosome. 00:41:54.600 --> 00:41:56.590 Volume, what is this volume? 00:42:01.147 --> 00:42:02.356 AUDIENCE: The elution volume. 00:42:02.356 --> 00:42:03.313 ELIZABETH NOLAN: Right. 00:42:03.313 --> 00:42:04.970 The volume eluted from the column. 00:42:04.970 --> 00:42:08.540 So looking at this trace, what do we see? 00:42:12.033 --> 00:42:15.526 AUDIENCE: There's a peak [INAUDIBLE] 00:42:15.526 --> 00:42:18.530 maybe there's some [INAUDIBLE] introduced later. 00:42:18.530 --> 00:42:19.600 ELIZABETH NOLAN: OK. 00:42:19.600 --> 00:42:22.610 So you've mixed what you see with an interpretation. 00:42:22.610 --> 00:42:25.932 So let's just stick right now to what do we see in the trace? 00:42:25.932 --> 00:42:27.640 And then we're going to think about where 00:42:27.640 --> 00:42:28.810 these things come from. 00:42:28.810 --> 00:42:31.630 And that's just something important, 00:42:31.630 --> 00:42:33.790 I think, with the problems we give in this course. 00:42:33.790 --> 00:42:37.050 First, you want to ask just what does the data say? 00:42:37.050 --> 00:42:39.280 And then, how do we interpret this data 00:42:39.280 --> 00:42:42.384 based on our knowledge of a system here, OK? 00:42:42.384 --> 00:42:44.800 So do you see what I mean, how you mixed what you see here 00:42:44.800 --> 00:42:47.050 and a potential interpretation? 00:42:47.050 --> 00:42:49.328 So just what do we see? 00:42:49.328 --> 00:42:50.462 AUDIENCE: Two peaks. 00:42:50.462 --> 00:42:51.670 ELIZABETH NOLAN: So there's-- 00:42:51.670 --> 00:42:52.169 Yeah. 00:42:52.169 --> 00:42:53.432 Rebecca? 00:42:53.432 --> 00:42:56.869 AUDIENCE: Just the large broad peak 00:42:56.869 --> 00:43:02.270 that results immediately, and then a smaller separate peak. 00:43:02.270 --> 00:43:04.640 ELIZABETH NOLAN: So that's a very nice description. 00:43:04.640 --> 00:43:09.680 We see a broad peak with high A260 absorbance, 00:43:09.680 --> 00:43:11.960 then it elutes immediately. 00:43:11.960 --> 00:43:15.890 And then later, there's a peak just after 30 mils that's 00:43:15.890 --> 00:43:17.150 sharper, right? 00:43:17.150 --> 00:43:18.725 So what are these peaks? 00:43:23.405 --> 00:43:24.790 What came off here? 00:43:31.552 --> 00:43:33.001 AUDIENCE: Everything else. 00:43:33.001 --> 00:43:33.970 ELIZABETH NOLAN: Yeah. 00:43:33.970 --> 00:43:35.400 So what's everything else? 00:43:40.190 --> 00:43:44.064 AUDIENCE: DNA, [INAUDIBLE]. 00:43:44.064 --> 00:43:44.980 ELIZABETH NOLAN: Yeah. 00:43:44.980 --> 00:43:47.320 So things that didn't stick to the column, right? 00:43:47.320 --> 00:43:48.390 They got washed out. 00:43:48.390 --> 00:43:51.700 So maybe the native ribosomes, right? 00:43:51.700 --> 00:43:54.700 Maybe there's DNA in there, tRNAs. 00:43:54.700 --> 00:43:57.940 Could there be EFTU with tRNA bound, right? 00:43:57.940 --> 00:44:03.100 And there are things we're not seeing, right? 00:44:03.100 --> 00:44:06.250 So what do we think about the second peak? 00:44:11.840 --> 00:44:13.230 AUDIENCE: It's much less broad. 00:44:13.230 --> 00:44:15.000 It's pretty sharp, so that will tell you 00:44:15.000 --> 00:44:18.835 that it's likely only one thing that 00:44:18.835 --> 00:44:20.621 has bound to the column a lot. 00:44:20.621 --> 00:44:22.245 So that would probably be your His-tag. 00:44:22.245 --> 00:44:23.924 ELIZABETH NOLAN: Is this a His-tag here? 00:44:23.924 --> 00:44:26.090 I know we're going back and forth because you're all 00:44:26.090 --> 00:44:27.256 more familiar with His-tags. 00:44:27.256 --> 00:44:28.912 AUDIENCE: Your affinity tag. 00:44:28.912 --> 00:44:29.870 ELIZABETH NOLAN: Right. 00:44:29.870 --> 00:44:32.990 So this is likely what was tagged, right? 00:44:32.990 --> 00:44:36.860 And with GSH elution, we disrupted 00:44:36.860 --> 00:44:38.600 the binding interaction with GST, 00:44:38.600 --> 00:44:40.190 and it came off the column. 00:44:40.190 --> 00:44:44.240 Do we know that it's only one thing? 00:44:44.240 --> 00:44:45.560 No. 00:44:45.560 --> 00:44:47.570 At this stage, we don't, right? 00:44:47.570 --> 00:44:50.515 So what are possibilities? 00:44:50.515 --> 00:44:52.850 What could this be? 00:44:52.850 --> 00:44:54.550 So we have the tag on the 50S. 00:45:04.142 --> 00:45:06.100 Because the only-- I mean, it's coming off here 00:45:06.100 --> 00:45:09.790 just based on the amount of GSH required to push it off 00:45:09.790 --> 00:45:10.856 the column there. 00:45:10.856 --> 00:45:13.231 AUDIENCE: It could be a mixture of intact ribosome, which 00:45:13.231 --> 00:45:14.832 is the cell that gets tagged. 00:45:14.832 --> 00:45:15.790 ELIZABETH NOLAN: Right. 00:45:15.790 --> 00:45:16.290 Yeah. 00:45:16.290 --> 00:45:19.090 So we don't know right now in terms of the whole composition 00:45:19.090 --> 00:45:20.740 of this peak, right? 00:45:20.740 --> 00:45:24.460 It could be intact 70S because 30S came down. 00:45:24.460 --> 00:45:26.970 It could be 50S alone. 00:45:26.970 --> 00:45:28.660 And there's always the possibility 00:45:28.660 --> 00:45:31.120 of some other contaminant that just for whatever reason 00:45:31.120 --> 00:45:33.830 came off the column then there. 00:45:33.830 --> 00:45:34.880 OK. 00:45:34.880 --> 00:45:37.000 Would you be excited by this chromatogram 00:45:37.000 --> 00:45:39.580 if you were the person at the bench doing this work? 00:45:44.283 --> 00:45:44.783 Yeah. 00:45:44.783 --> 00:45:47.030 You'd be super excited, right? 00:45:47.030 --> 00:45:50.320 So it looks quite good. 00:45:50.320 --> 00:45:53.090 So of course, there needs to be some more analyses done. 00:45:53.090 --> 00:45:55.820 And one analysis we're not going to go into in detail 00:45:55.820 --> 00:45:59.630 but they needed to ask, is this really all tagged ribosome 00:45:59.630 --> 00:46:02.180 or is there also some contamination of wild-type? 00:46:02.180 --> 00:46:06.530 And so they designed some analysis using a technique 00:46:06.530 --> 00:46:09.380 called primary extension to look at that there. 00:46:09.380 --> 00:46:12.080 And what they saw is that they primarily 00:46:12.080 --> 00:46:15.480 had the tagged ribosome, which was good news. 00:46:15.480 --> 00:46:17.780 So getting at the question in terms of what's actually 00:46:17.780 --> 00:46:21.050 in this peak, they looked at effectively, 00:46:21.050 --> 00:46:24.095 say, subunit integrity. 00:46:24.095 --> 00:46:25.880 And how did they do this? 00:46:25.880 --> 00:46:28.492 They used centrifugation. 00:46:28.492 --> 00:46:31.640 And does anyone recall what type of centrifugation they used? 00:46:36.262 --> 00:46:37.720 AUDIENCE: Sucrose density gradient. 00:46:37.720 --> 00:46:38.595 ELIZABETH NOLAN: Yes. 00:46:38.595 --> 00:46:41.120 So they used a sucrose gradient, right? 00:46:41.120 --> 00:46:43.696 And how does that let you do separation? 00:46:43.696 --> 00:46:44.602 AUDIENCE: By density. 00:46:44.602 --> 00:46:46.060 ELIZABETH NOLAN: Right, by density. 00:46:46.060 --> 00:46:50.090 And we have the different subunits of different sizes, 00:46:50.090 --> 00:46:52.060 different density there, right? 00:46:52.060 --> 00:46:56.780 So this is the data they show. 00:46:56.780 --> 00:47:00.490 And so, again, we want to look at these data and ask 00:47:00.490 --> 00:47:03.520 what do we see and what does that tell us, right? 00:47:03.520 --> 00:47:07.120 So these are the results from the sucrose gradient 00:47:07.120 --> 00:47:08.470 centrifugation. 00:47:08.470 --> 00:47:12.920 And they looked at untied wild-type ribosomes, 00:47:12.920 --> 00:47:16.810 so isolated 70S and then the tied ribosome. 00:47:16.810 --> 00:47:20.680 So what was the key part of the sample preparation here? 00:47:23.200 --> 00:47:25.090 How did they prepare these samples 00:47:25.090 --> 00:47:27.820 to be able to look at the subunits individually? 00:47:33.199 --> 00:47:34.990 Because the question they're getting at is, 00:47:34.990 --> 00:47:39.520 what is the ratio of the 50S to the 30S in the sample that 00:47:39.520 --> 00:47:42.912 was purified from the column? 00:47:42.912 --> 00:47:47.374 AUDIENCE: They dialyzed using magnesium buffer. 00:47:47.374 --> 00:47:48.290 ELIZABETH NOLAN: Yeah. 00:47:48.290 --> 00:47:50.690 So what was it about the magnesium in the buffer? 00:47:54.022 --> 00:47:55.926 AUDIENCE: It causes disassociation 00:47:55.926 --> 00:47:57.840 between the 30S and the 50S. 00:47:57.840 --> 00:47:58.862 ELIZABETH NOLAN: So why? 00:47:58.862 --> 00:48:00.570 Maybe you said it, and I didn't hear you. 00:48:00.570 --> 00:48:03.969 What was it about the buffer that allowed this? 00:48:03.969 --> 00:48:07.079 AUDIENCE: Is it the positive charge? 00:48:07.079 --> 00:48:09.495 ELIZABETH NOLAN: Well, it is the magnesium, but was it low 00:48:09.495 --> 00:48:10.560 or high magnesium buffer? 00:48:10.560 --> 00:48:11.310 AUDIENCE: Oh, low. 00:48:11.310 --> 00:48:12.630 ELIZABETH NOLAN: Low, Right? 00:48:12.630 --> 00:48:15.960 So we learned that magnesium is important for interactions 00:48:15.960 --> 00:48:17.530 between these subunits, right? 00:48:17.530 --> 00:48:20.430 And you've seen some of the experimental details 00:48:20.430 --> 00:48:22.800 in the paper for recitation two and three, 00:48:22.800 --> 00:48:25.530 they used different concentrations of magnesium. 00:48:25.530 --> 00:48:28.140 In this case, they want to separate the two subunits. 00:48:28.140 --> 00:48:32.340 So they basically used a low magnesium buffer 00:48:32.340 --> 00:48:36.570 to allow them to dissociate. 00:48:36.570 --> 00:48:39.720 So in these data, what are we looking at? 00:48:39.720 --> 00:48:41.670 The axes aren't shown, but what are they? 00:48:46.320 --> 00:48:47.715 AUDIENCE: I have a question. 00:48:47.715 --> 00:48:50.046 Why not just a no-magnesium buffer? 00:48:55.730 --> 00:48:58.950 ELIZABETH NOLAN: Could that be bad for the sample? 00:48:58.950 --> 00:49:01.629 How low is the magnesium in the buffer? 00:49:01.629 --> 00:49:02.670 AUDIENCE: One millimolar. 00:49:02.670 --> 00:49:04.260 ELIZABETH NOLAN: One millimolar. 00:49:04.260 --> 00:49:08.060 So where else might the magnesium go? 00:49:08.060 --> 00:49:10.353 Is it only important for this interaction? 00:49:20.644 --> 00:49:22.185 AUDIENCE: I mean I guess it's useful. 00:49:22.185 --> 00:49:23.976 It can be used in a lot of different places 00:49:23.976 --> 00:49:25.197 actually in the cell. 00:49:25.197 --> 00:49:26.030 ELIZABETH NOLAN: OK. 00:49:26.030 --> 00:49:27.540 But we don't have the cell here. 00:49:27.540 --> 00:49:29.174 We have the purified ribosome. 00:49:29.174 --> 00:49:30.590 AUDIENCE: Is it possible that it's 00:49:30.590 --> 00:49:32.820 holding together the actual conformation 00:49:32.820 --> 00:49:35.032 of the 50S and the 30S? 00:49:35.032 --> 00:49:36.240 ELIZABETH NOLAN: Yeah, right. 00:49:36.240 --> 00:49:37.375 I mean, that may-- 00:49:37.375 --> 00:49:39.000 I actually don't know what would happen 00:49:39.000 --> 00:49:41.460 if this goes into a no-magnesium buffer, right? 00:49:41.460 --> 00:49:44.490 But I think there's about a dozen contacts where 00:49:44.490 --> 00:49:46.614 magnesium is used between the two subunits. 00:49:46.614 --> 00:49:48.030 And you can imagine there's plenty 00:49:48.030 --> 00:49:50.190 of interactions of magnesium or cations 00:49:50.190 --> 00:49:52.121 in other places of each subunit. 00:49:52.121 --> 00:49:54.620 Joanne, do you happen to know what happens if the ribosome-- 00:49:54.620 --> 00:49:56.335 I think you get a big unfolded mess. 00:49:56.335 --> 00:49:58.793 JOANNE STUBBE: It would be hard to get rid of the magnesium 00:49:58.793 --> 00:50:01.477 because it has key binding sites along the place 00:50:01.477 --> 00:50:05.330 where it comes off and goes back on. 00:50:05.330 --> 00:50:06.630 ELIZABETH NOLAN: Yeah. 00:50:06.630 --> 00:50:08.100 So what are the axes? 00:50:08.100 --> 00:50:10.100 If we were to have them, what are we looking at? 00:50:13.022 --> 00:50:15.424 AUDIENCE: I think the gradient. 00:50:15.424 --> 00:50:16.340 ELIZABETH NOLAN: Yeah. 00:50:16.340 --> 00:50:16.839 Right. 00:50:16.839 --> 00:50:19.550 So we have basically, yeah, the percent sucrose, 00:50:19.550 --> 00:50:22.730 say, the gradient-- or the density, right? 00:50:22.730 --> 00:50:25.670 And how are we seeing these peaks? 00:50:35.800 --> 00:50:40.700 AUDIENCE: We see two peaks and the upper graph 00:50:40.700 --> 00:50:46.250 we see the peaks are more similar in size. 00:50:46.250 --> 00:50:51.150 And then in the second part we see-- well, we see two peaks 00:50:51.150 --> 00:50:53.600 but one peak has a shift over. 00:50:53.600 --> 00:50:55.640 ELIZABETH NOLAN: Right. 00:50:55.640 --> 00:50:57.470 So just backing up. 00:50:57.470 --> 00:50:59.725 That's all certainly the case. 00:50:59.725 --> 00:51:02.060 How are we detecting these peaks? 00:51:02.060 --> 00:51:03.140 What's the readout? 00:51:03.140 --> 00:51:07.200 So we have some sucrose gradient here. 00:51:07.200 --> 00:51:09.057 How do we see the peaks? 00:51:09.057 --> 00:51:10.370 AUDIENCE: PUB. 00:51:10.370 --> 00:51:12.400 ELIZABETH NOLAN: Well, what, more specifically? 00:51:12.400 --> 00:51:13.710 AUDIENCE: Oh, A280. 00:51:13.710 --> 00:51:14.626 ELIZABETH NOLAN: Yeah. 00:51:14.626 --> 00:51:17.660 So here, they're using the absorbance at 280, right? 00:51:17.660 --> 00:51:23.611 So a little different than the chromatogram, but that's OK, 00:51:23.611 --> 00:51:24.110 right? 00:51:24.110 --> 00:51:28.025 There's 280 absorbance here and maybe their instrument for this 00:51:28.025 --> 00:51:29.820 didn't allow 260. 00:51:29.820 --> 00:51:36.660 So as we just heard, if we look at the untagged ribosome, 00:51:36.660 --> 00:51:40.380 we see that there's two peaks, and they're nicely labeled. 00:51:40.380 --> 00:51:43.340 It's nicely labeled with the 30S here 00:51:43.340 --> 00:51:49.021 in terms of percent sucrose in the gradient and 50S here, 00:51:49.021 --> 00:51:49.520 right? 00:51:49.520 --> 00:51:53.360 And so we see kind of in our standard control, 00:51:53.360 --> 00:51:56.180 the peak for the 30S is smaller than the 50S, 00:51:56.180 --> 00:51:57.710 and this is where they're placed. 00:51:57.710 --> 00:52:02.120 And so what we want to do is compare this data to the data 00:52:02.120 --> 00:52:04.020 here for the tagged ribosome. 00:52:04.020 --> 00:52:04.520 OK. 00:52:04.520 --> 00:52:07.670 So what are the two major observations, 00:52:07.670 --> 00:52:09.237 two major differences? 00:52:12.646 --> 00:52:14.594 Rebecca. 00:52:14.594 --> 00:52:18.003 AUDIENCE: The second one's enriched for the proteasome, 00:52:18.003 --> 00:52:20.940 there's relatively higher concentration of 50S. 00:52:20.940 --> 00:52:22.230 ELIZABETH NOLAN: OK and-- 00:52:22.230 --> 00:52:24.376 AUDIENCE: And it's [INAUDIBLE]. 00:52:24.376 --> 00:52:25.250 ELIZABETH NOLAN: Yes. 00:52:25.250 --> 00:52:27.480 So or another way to say that, maybe, it's 00:52:27.480 --> 00:52:30.570 depleted in the 30S, right? 00:52:30.570 --> 00:52:33.870 But I think you're coming across the same observation, right? 00:52:33.870 --> 00:52:39.559 So first we see there is a peak corresponding to the 30S 00:52:39.559 --> 00:52:41.100 and then there's this peak here which 00:52:41.100 --> 00:52:42.780 is shifted relative to what we saw 00:52:42.780 --> 00:52:45.240 for the 50S and the untagged ribosome. 00:52:45.240 --> 00:52:47.440 And the peak heights or peak areas, 00:52:47.440 --> 00:52:49.880 however you want to analyze it, that ratio 00:52:49.880 --> 00:52:53.170 is very different than what we see up here. 00:52:53.170 --> 00:52:56.990 So why is this shifted and why is there more 50S? 00:53:00.670 --> 00:53:02.090 AUDIENCE: Can I ask a question? 00:53:02.090 --> 00:53:05.990 So can you definitively say that it's enriched or depleted 00:53:05.990 --> 00:53:08.050 in 30S? 00:53:08.050 --> 00:53:10.690 I don't know a lot about biochemistry in general, 00:53:10.690 --> 00:53:17.010 but can you say that possibly the multiple histamine-- 00:53:17.010 --> 00:53:18.686 oh, it's not histamine. 00:53:21.711 --> 00:53:24.470 AUDIENCE: Is it just tagged because of the shift. 00:53:24.470 --> 00:53:26.750 I mean, it seems like such a small derivation. 00:53:26.750 --> 00:53:27.583 ELIZABETH NOLAN: OK. 00:53:27.583 --> 00:53:30.200 So what else is there in the sample? 00:53:30.200 --> 00:53:33.400 So we have that stem loop tag, but based on how they eluted 00:53:33.400 --> 00:53:35.410 this, what else is there? 00:53:35.410 --> 00:53:36.310 AUDIENCE: The fusion. 00:53:36.310 --> 00:53:38.250 ELIZABETH NOLAN: The fusion protein, right? 00:53:38.250 --> 00:53:40.000 So then the question is-- and this is just 00:53:40.000 --> 00:53:42.416 getting to the point of needing to look at all the details 00:53:42.416 --> 00:53:45.070 in terms of what's done in someone's experiment to try 00:53:45.070 --> 00:53:47.470 to figure out what's happening. 00:53:47.470 --> 00:53:51.730 Is the MS2 coat protein alone enough to cause a shift? 00:53:54.250 --> 00:53:55.810 So what else might that be? 00:54:02.572 --> 00:54:06.436 AUDIENCE: Could it be the GST? 00:54:06.436 --> 00:54:09.680 ELIZABETH NOLAN: Well, you know that the GST/MS2 is there 00:54:09.680 --> 00:54:14.030 because it was GSH that was used to elute that whole thing. 00:54:14.030 --> 00:54:18.890 So the ribosome and the GST/MS2 fusion. 00:54:18.890 --> 00:54:22.130 So I guess the question is, just thinking what is 00:54:22.130 --> 00:54:23.570 the size of the fusion protein? 00:54:26.270 --> 00:54:28.670 And then how does that compare to the ribosome? 00:54:28.670 --> 00:54:30.710 And what does that mean in terms of a shift 00:54:30.710 --> 00:54:32.750 to a different percent sucrose? 00:54:38.306 --> 00:54:41.380 So just what are possibilities? 00:54:41.380 --> 00:54:45.220 We learned that the fusion protein had some problems, 00:54:45.220 --> 00:54:47.110 that it liked to aggregate. 00:54:47.110 --> 00:54:49.180 Is it possible that contributes? 00:54:49.180 --> 00:54:53.700 Is it possible that not all of the 70S was dissociated, right? 00:54:53.700 --> 00:54:56.680 So there is not a label for where the 70S would come here. 00:54:56.680 --> 00:54:59.980 These are just things to think about when looking at the data 00:54:59.980 --> 00:55:04.060 here and to look at their explanation. 00:55:04.060 --> 00:55:06.430 So is this good news or bad news? 00:55:06.430 --> 00:55:08.524 And is there a solution? 00:55:08.524 --> 00:55:09.289 Yeah? 00:55:09.289 --> 00:55:09.955 AUDIENCE: Sorry. 00:55:09.955 --> 00:55:10.909 I had a question. 00:55:10.909 --> 00:55:14.725 Since they're monitoring the A280, so the protein 00:55:14.725 --> 00:55:21.005 absorption, since the 50S would have the fusion 00:55:21.005 --> 00:55:22.380 protein associated with it still, 00:55:22.380 --> 00:55:25.170 wouldn't that affect the A280? 00:55:25.170 --> 00:55:27.120 Can we definitively say that it's 00:55:27.120 --> 00:55:29.550 actually depleted in the 30S or is maybe 00:55:29.550 --> 00:55:33.430 just the absorbance of the 50S higher for the tagged protein 00:55:33.430 --> 00:55:34.347 because of the fusion? 00:55:34.347 --> 00:55:35.263 ELIZABETH NOLAN: Yeah. 00:55:35.263 --> 00:55:36.660 So that's a good point, right? 00:55:36.660 --> 00:55:40.230 The fusion protein is going to have some absorbance defined 00:55:40.230 --> 00:55:42.050 by its extinction coefficient. 00:55:42.050 --> 00:55:45.030 And then the question is, what is that in magnitude? 00:55:45.030 --> 00:55:47.520 And how does that compare to, say, the total extinction 00:55:47.520 --> 00:55:49.860 coefficient of the 50S, right? 00:55:49.860 --> 00:55:54.012 So there are a number of ribosomal proteins, 20 to 30, 00:55:54.012 --> 00:55:55.470 and we don't know how many of those 00:55:55.470 --> 00:56:00.110 got through this purification, but they're there. 00:56:00.110 --> 00:56:02.610 So that's how I would go about analyzing that. 00:56:02.610 --> 00:56:05.430 I actually don't know what the relative absorbance 00:56:05.430 --> 00:56:11.330 of the fusion protein to the intact 50S ribosome is 00:56:11.330 --> 00:56:15.088 but that's a caveat to consider and could contribute. 00:56:20.360 --> 00:56:21.547 So what do we think? 00:56:21.547 --> 00:56:23.130 What are you going to do for an assay? 00:56:36.740 --> 00:56:39.490 If we were to set these ribosomes up, the wild type 00:56:39.490 --> 00:56:42.400 and the mutant one, and, let's say, do a peptide bond 00:56:42.400 --> 00:56:46.770 formation reaction, like what they presented in the paper 00:56:46.770 --> 00:56:48.680 as is, what would you expect? 00:56:48.680 --> 00:56:51.160 Are you going to see the same activity or less activity? 00:56:55.160 --> 00:56:59.230 Working under the model that we don't have enough 30S here 00:56:59.230 --> 00:57:04.550 to fully form 70S ribosomes. 00:57:04.550 --> 00:57:07.100 AUDIENCE: You'd expect the 30S to act 00:57:07.100 --> 00:57:09.970 like the limiting agent in peptide bond formation 00:57:09.970 --> 00:57:11.240 where they come together. 00:57:11.240 --> 00:57:15.790 And you can only make as much ribosome as you have 30S. 00:57:15.790 --> 00:57:19.222 So you would get less peptide bond formation. 00:57:19.222 --> 00:57:20.180 ELIZABETH NOLAN: Right. 00:57:20.180 --> 00:57:23.240 So you'd have fewer 70S assembled ribosomes 00:57:23.240 --> 00:57:25.141 that can do translation. 00:57:25.141 --> 00:57:25.640 Right. 00:57:25.640 --> 00:57:27.501 So what was their solution to this problem? 00:57:31.349 --> 00:57:33.190 Or if you don't remember, what would you do? 00:57:50.800 --> 00:57:53.770 What happens if we don't have enough of a given component? 00:57:53.770 --> 00:57:57.890 We'll finish on this note. 00:57:57.890 --> 00:58:00.160 AUDIENCE: You just add more of the 30S. 00:58:00.160 --> 00:58:02.290 ELIZABETH NOLAN: Yeah, right? 00:58:02.290 --> 00:58:05.990 So you can imagine adding in more 30s there. 00:58:05.990 --> 00:58:09.100 We know how to purify wild-type ribosomes, 00:58:09.100 --> 00:58:11.140 can dissociate the subunits and imagine 00:58:11.140 --> 00:58:15.460 purifying 30S and adding that into the reaction to have 00:58:15.460 --> 00:58:17.920 the correct stochiometry there. 00:58:17.920 --> 00:58:21.340 So that's exactly what they did in their assays. 00:58:21.340 --> 00:58:24.580 And so I encourage you to take a look at the assays 00:58:24.580 --> 00:58:27.040 they did for peptide bond formation 00:58:27.040 --> 00:58:29.500 and for peptide release. 00:58:29.500 --> 00:58:33.610 And in the posted notes, there's a schematic overview 00:58:33.610 --> 00:58:36.310 of everything that was done to get the components 00:58:36.310 --> 00:58:37.330 of their syringes. 00:58:37.330 --> 00:58:39.070 So these were quench flow experiments, 00:58:39.070 --> 00:58:41.470 like what you talked about in recitation in prior weeks 00:58:41.470 --> 00:58:43.570 and we talked about with EFP. 00:58:43.570 --> 00:58:46.630 And there's also an example where they used puromycin, 00:58:46.630 --> 00:58:51.820 like what we saw with EFP, in the experimental setup, so 00:58:51.820 --> 00:58:55.710 that antibiotic that causes change termination there. 00:58:55.710 --> 00:58:58.990 And so what they found is that these tagged ribosomes really 00:58:58.990 --> 00:59:04.190 work quite well in terms of the kinetic comparison there. 00:59:04.190 --> 00:59:06.040 And they moved on with this methodology 00:59:06.040 --> 00:59:08.910 to use it to purify new ribosomes for other studies. 00:59:08.910 --> 00:59:12.370 So it turned out to be a useful method for their lab, 00:59:12.370 --> 00:59:15.070 and I would argue, what would be a useful method for other labs, 00:59:15.070 --> 00:59:19.570 based on the information they presented in their paper there. 00:59:19.570 --> 00:59:20.740 OK? 00:59:20.740 --> 00:59:24.520 So you're off the hook, and I'll see you in recitation 00:59:24.520 --> 00:59:26.670 next week.