1 00:00:00,500 --> 00:00:02,640 The following content is provided under a Creative 2 00:00:02,640 --> 00:00:04,210 Commons license. 3 00:00:04,210 --> 00:00:06,510 Your support will help MIT Open Courseware 4 00:00:06,510 --> 00:00:10,840 continue to offer high quality educational resources for free. 5 00:00:10,840 --> 00:00:13,500 To make a donation or view additional materials 6 00:00:13,500 --> 00:00:17,440 from hundreds of MIT courses, visit MIT Open Courseware 7 00:00:17,440 --> 00:00:18,410 at ocw.mit.edu. 8 00:00:25,173 --> 00:00:26,840 ELIZABETH NOLAN: We're going to continue 9 00:00:26,840 --> 00:00:29,690 where we left off last time. 10 00:00:29,690 --> 00:00:33,470 So briefly I'll make a few points about initiation 11 00:00:33,470 --> 00:00:35,487 of translation in prokaryotes. 12 00:00:35,487 --> 00:00:37,820 And then where we're going to spend the bulk of the time 13 00:00:37,820 --> 00:00:42,380 today is with a review of tRNAs and then discussing 14 00:00:42,380 --> 00:00:44,980 the aminoacyl-tRNA synthetases, which 15 00:00:44,980 --> 00:00:48,020 are the enzymes responsible for loading 16 00:00:48,020 --> 00:00:51,600 amino acids onto the three prime end of the tRNA. 17 00:00:51,600 --> 00:00:55,100 And these points are important because these process has 18 00:00:55,100 --> 00:00:57,350 to happen in order for the amino acids 19 00:00:57,350 --> 00:00:59,750 to be delivered to the ribosome, which 20 00:00:59,750 --> 00:01:02,430 is where we'll go on Wednesday. 21 00:01:02,430 --> 00:01:07,970 So the first questions are, how does initiation happen? 22 00:01:07,970 --> 00:01:12,020 So how does this ribosome, 70S ribosome, 23 00:01:12,020 --> 00:01:16,280 get assembled with the mRNA and initiator tRNA bound? 24 00:01:16,280 --> 00:01:17,990 And then we're going to ask, how do we 25 00:01:17,990 --> 00:01:21,980 get an aminoacyl-tRNA, such that the amino acids can 26 00:01:21,980 --> 00:01:24,770 be delivered to the ribosome? 27 00:01:24,770 --> 00:01:29,600 So first, for initiation in prokaryotes, 28 00:01:29,600 --> 00:01:31,640 there's a few steps to this process. 29 00:01:31,640 --> 00:01:34,700 We'll just look at these at a basically superficial level 30 00:01:34,700 --> 00:01:35,630 of detail. 31 00:01:35,630 --> 00:01:39,110 But recall that there are translation factors. 32 00:01:39,110 --> 00:01:42,610 And during initiation, there are three initiation factors-- 33 00:01:42,610 --> 00:01:45,020 so IF 1, 2, and 3-- 34 00:01:45,020 --> 00:01:50,000 that are required to help assemble the 70S ribosome here. 35 00:01:50,000 --> 00:01:54,800 So first in terms of initiation, what happens 36 00:01:54,800 --> 00:02:01,650 is that the mRNA needs to bind to the 16S RNA of the 30S 37 00:02:01,650 --> 00:02:02,150 subunit. 38 00:02:10,100 --> 00:02:21,560 And so I point this out because at this stage in the process, 39 00:02:21,560 --> 00:02:24,350 the 70S ribosome isn't assembled yet. 40 00:02:24,350 --> 00:02:29,600 So we have the mRNA binding to the small subunit. 41 00:02:29,600 --> 00:02:37,820 And this process requires initiation factor 3. 42 00:02:37,820 --> 00:02:41,600 And effectively what happens is that the mRNA has 43 00:02:41,600 --> 00:02:44,000 a region called the Shine-Dalgarno sequence 44 00:02:44,000 --> 00:02:47,300 in prokaryotes, which is the site of ribosome binding. 45 00:02:47,300 --> 00:02:49,490 And then upstream of that is a start codon 46 00:02:49,490 --> 00:02:52,080 that signals for the start of translation. 47 00:02:52,080 --> 00:02:58,290 So if we think about the mRNA of the five prime end, 48 00:02:58,290 --> 00:03:06,550 and somewhere there's a sequence that 49 00:03:06,550 --> 00:03:07,990 signals for ribosome binding. 50 00:03:15,540 --> 00:03:21,790 OK, and then we have our start codon 51 00:03:21,790 --> 00:03:23,860 that signals the start of translation. 52 00:03:27,890 --> 00:03:30,360 OK. 53 00:03:30,360 --> 00:03:36,620 And so this gets translated here. 54 00:03:36,620 --> 00:03:40,640 OK, so this start codon pairs with initiator tRNA. 55 00:03:47,360 --> 00:03:50,090 And this initiator tRNA is special. 56 00:03:50,090 --> 00:03:53,930 One reason why it's special is because the amino acid attached 57 00:03:53,930 --> 00:03:56,825 is an N-Formylmethionine OK. 58 00:04:13,420 --> 00:04:20,079 So sometimes the initiator tRNA is called f-met tRNA f-met 59 00:04:20,079 --> 00:04:23,650 as an abbreviation there. 60 00:04:23,650 --> 00:04:27,610 So just as some overview here, what 61 00:04:27,610 --> 00:04:29,440 we're seeing in this alignment is 62 00:04:29,440 --> 00:04:31,750 a number of the ribosome binding sites, 63 00:04:31,750 --> 00:04:35,230 or Shine-Dalgarno sequences in prokaryotes. 64 00:04:35,230 --> 00:04:39,400 We have the start codon on that pairs with the initiator tRNA. 65 00:04:39,400 --> 00:04:43,330 And here's a schematic depiction of what I've indicated here 66 00:04:43,330 --> 00:04:44,800 on the board. 67 00:04:44,800 --> 00:04:50,020 OK, so the mRNA binds to the 16S of the 30S subunit. 68 00:04:50,020 --> 00:04:53,230 So the 70S is not assembled at this stage. 69 00:04:53,230 --> 00:04:56,360 And IF3 is involved, as I said. 70 00:04:56,360 --> 00:05:00,550 The Shine-Dalgarno sequence determines the start site. 71 00:05:00,550 --> 00:05:03,330 And we determine the reading frame, as well. 72 00:05:03,330 --> 00:05:07,210 So here is just an indicating translation of a polypeptide. 73 00:05:07,210 --> 00:05:10,660 What happens after that? 74 00:05:10,660 --> 00:05:16,360 So after that, it's necessary to assemble the 70S ribosome, 75 00:05:16,360 --> 00:05:19,510 have the initiator tRNA in the P site, 76 00:05:19,510 --> 00:05:22,630 and have the cell ready to go for translation. 77 00:05:22,630 --> 00:05:25,450 And here's just one cartoon overview 78 00:05:25,450 --> 00:05:28,600 that we'll use as a description of this process. 79 00:05:28,600 --> 00:05:29,710 OK. 80 00:05:29,710 --> 00:05:31,210 So what do we see? 81 00:05:31,210 --> 00:05:33,520 We've talked about this step so far. 82 00:05:33,520 --> 00:05:37,190 We see there's a role for initiation factor 1. 83 00:05:37,190 --> 00:05:40,570 And in this cartoon, if we imagine the E site, 84 00:05:40,570 --> 00:05:44,050 the P site, and the A site, what we see 85 00:05:44,050 --> 00:05:48,160 is that IF1 is binding to the site of the ribosome. 86 00:05:48,160 --> 00:05:49,930 And one way we can think about this 87 00:05:49,930 --> 00:05:53,930 is that the initiator tRNA has to get to the P site. 88 00:05:53,930 --> 00:05:57,460 And so that region is blocked to facilitate the initiator 89 00:05:57,460 --> 00:05:59,940 tRNA getting to the P site. 90 00:05:59,940 --> 00:06:04,630 OK, we see that initiator tRNA binding to the P site. 91 00:06:04,630 --> 00:06:07,600 And this happens via formation of a ternary complex 92 00:06:07,600 --> 00:06:10,600 with IF2 and GTP. 93 00:06:10,600 --> 00:06:15,640 So initiation factor 2 hydrolyzes GTP. 94 00:06:15,640 --> 00:06:18,910 There's an event that results in joining of the two subunits. 95 00:06:18,910 --> 00:06:21,250 And there has to be dissociation of these initiation 96 00:06:21,250 --> 00:06:24,550 factors for the ribosome to be ready to accept 97 00:06:24,550 --> 00:06:28,090 its first aminoacyl-tRNA in the A site. 98 00:06:28,090 --> 00:06:31,120 OK, so the outcome of this process 99 00:06:31,120 --> 00:06:33,370 here is that we have an assembled 70S 100 00:06:33,370 --> 00:06:36,880 ribosome with the initiator tRNA in the P site. 101 00:06:36,880 --> 00:06:40,270 The A site is empty, so it can accommodate 102 00:06:40,270 --> 00:06:43,300 an incoming aminoacyl-tRNA. 103 00:06:43,300 --> 00:06:47,120 And the E site or exit site is also empty. 104 00:06:47,120 --> 00:06:49,840 So that's the main take home for initiation. 105 00:06:49,840 --> 00:06:51,340 And that's the extent to which we're 106 00:06:51,340 --> 00:06:54,770 going to discuss it within this class. 107 00:06:54,770 --> 00:06:57,520 So in order to get to the elongation cycle, 108 00:06:57,520 --> 00:07:01,200 we need to get the aminoacyl-tRNA into the A site. 109 00:07:01,200 --> 00:07:05,410 And that's going to require the help of EF-Tu, so elongation 110 00:07:05,410 --> 00:07:07,630 factor Tu. 111 00:07:07,630 --> 00:07:10,660 Before we discuss how elongation factor 112 00:07:10,660 --> 00:07:14,240 Tu is going to help deliver that aminoacyl-tRNA, 113 00:07:14,240 --> 00:07:16,810 we need to talk about how we get the aminoacyl-tRNA 114 00:07:16,810 --> 00:07:18,230 in the first place. 115 00:07:18,230 --> 00:07:21,610 So what is the tRNA structure, just as a review 116 00:07:21,610 --> 00:07:23,810 to get everyone up to speed. 117 00:07:23,810 --> 00:07:27,730 How are amino acid monomers attached to the tRNA? 118 00:07:27,730 --> 00:07:30,080 And how is the correct amino acid attached? 119 00:07:30,080 --> 00:07:31,780 So this is an aspect of fidelity, 120 00:07:31,780 --> 00:07:36,040 which came up as a concept last week in lecture. 121 00:07:36,040 --> 00:07:37,540 And so we'll look at the mechanism 122 00:07:37,540 --> 00:07:40,180 of aminoacyl-tRNA synthetases to see 123 00:07:40,180 --> 00:07:42,760 how is the correct amino acid attached, 124 00:07:42,760 --> 00:07:46,450 and then what happens if the wrong amino acid is selected. 125 00:07:46,450 --> 00:07:48,400 Are there mechanisms to correct that? 126 00:07:48,400 --> 00:07:52,430 And if it's not corrected, what are the consequences here? 127 00:07:52,430 --> 00:07:59,900 So moving forward with that, we're 128 00:07:59,900 --> 00:08:05,340 going to focus on the tRNAs and addressing those questions. 129 00:08:05,340 --> 00:08:09,260 So just as a review, so we can think 130 00:08:09,260 --> 00:08:18,990 about tRNA secondary structure, which is often 131 00:08:18,990 --> 00:08:20,430 described as cloverleaf. 132 00:08:32,049 --> 00:08:33,620 So we have a five prime end. 133 00:08:37,110 --> 00:08:38,970 The tRNA has several arms. 134 00:08:53,750 --> 00:08:54,470 OK. 135 00:08:54,470 --> 00:08:55,680 So we have a D arm. 136 00:08:58,430 --> 00:09:01,610 This arm here has the anticodon that pairs 137 00:09:01,610 --> 00:09:02,850 with the codon of the mRNA. 138 00:09:08,310 --> 00:09:18,550 We have a variable arm, this arm here. 139 00:09:18,550 --> 00:09:24,190 And we have this three prime end here, 140 00:09:24,190 --> 00:09:27,860 where the amino acids get attached. 141 00:09:27,860 --> 00:09:30,640 So this, in terms of base numbering, 142 00:09:30,640 --> 00:09:36,370 we have C74, C75, A76 here. 143 00:09:38,780 --> 00:09:39,280 OH. 144 00:09:39,280 --> 00:09:42,415 This is often called the CCA acceptor stem. 145 00:09:48,130 --> 00:09:50,470 And the amino acids are attached here. 146 00:09:50,470 --> 00:09:51,970 I'm going to abbreviate amino acid 147 00:09:51,970 --> 00:10:00,935 as AA via an ester linkage. 148 00:10:05,820 --> 00:10:08,470 And these ester linkages are important for the chemistry 149 00:10:08,470 --> 00:10:10,490 that happens in the ribosome. 150 00:10:10,490 --> 00:10:10,990 OK. 151 00:10:10,990 --> 00:10:16,570 So we can imagine just if we have abbreviating the tRNA 152 00:10:16,570 --> 00:10:24,110 structure like this and if we think about the sugar of A76-- 153 00:10:32,370 --> 00:10:32,910 bless you. 154 00:10:35,660 --> 00:10:37,140 OK. 155 00:10:37,140 --> 00:10:44,580 We have one prime, two prime, three prime here. 156 00:10:44,580 --> 00:10:47,370 This type of connectivity here. 157 00:10:47,370 --> 00:10:51,652 And this is abbreviated throughout as amino acid tRNA, 158 00:10:51,652 --> 00:10:56,730 aa in general terms, or the three-letter abbreviations, 159 00:10:56,730 --> 00:11:01,980 like what we saw for f-met tRNA f-met with the initiator tRNA 160 00:11:01,980 --> 00:11:02,480 here. 161 00:11:07,210 --> 00:11:10,860 So here's a schematic of a tRNA secondary structure with a bit 162 00:11:10,860 --> 00:11:13,140 more detail than what I show you on the board. 163 00:11:15,960 --> 00:11:17,880 And something we need to keep in mind 164 00:11:17,880 --> 00:11:22,020 is even though we often draw the tRNA in this cloverleaf type 165 00:11:22,020 --> 00:11:24,660 depiction, it has tertiary structure. 166 00:11:24,660 --> 00:11:27,660 And so it's very important to think about this structure 167 00:11:27,660 --> 00:11:31,830 as we think about how the tRNAs enter the various sites 168 00:11:31,830 --> 00:11:33,160 of the ribosome. 169 00:11:33,160 --> 00:11:33,660 OK. 170 00:11:33,660 --> 00:11:36,540 So this structure is L-shaped. 171 00:11:36,540 --> 00:11:39,780 And I like this depiction here because regions 172 00:11:39,780 --> 00:11:41,580 of the secondary structure are color 173 00:11:41,580 --> 00:11:44,280 coded with the corresponding regions 174 00:11:44,280 --> 00:11:46,820 of this tertiary structure here. 175 00:11:46,820 --> 00:11:49,380 OK, so we see the shell shape of an L, 176 00:11:49,380 --> 00:11:53,370 rather upside down here, where we have the CCA acceptor stem 177 00:11:53,370 --> 00:12:01,720 over here and the anticodon arm and anticodon region down here. 178 00:12:01,720 --> 00:12:06,900 So what is a consequence of this structure? 179 00:12:06,900 --> 00:12:09,690 The tRNA is quite narrow. 180 00:12:09,690 --> 00:12:15,070 So we're thinking about 20 to 25 Angstroms in width. 181 00:12:15,070 --> 00:12:18,540 And if we think about this in the context of the ribosome 182 00:12:18,540 --> 00:12:21,210 and the peptidyl transferase center, 183 00:12:21,210 --> 00:12:24,930 3 tRNAs need to fit into that catalytic center 184 00:12:24,930 --> 00:12:26,970 during the elongation cycle. 185 00:12:26,970 --> 00:12:30,060 So it makes sense that they're relatively narrow. 186 00:12:30,060 --> 00:12:33,660 This allows three to fit there. 187 00:12:33,660 --> 00:12:37,350 So as we think about the translation process 188 00:12:37,350 --> 00:12:40,110 and also think about some of the translation factors, 189 00:12:40,110 --> 00:12:43,320 we want to keep this type of structure in mind here. 190 00:12:47,820 --> 00:12:51,540 Here's just another view of that, 191 00:12:51,540 --> 00:12:53,610 with some additional descriptions 192 00:12:53,610 --> 00:12:55,530 of the overall structure. 193 00:12:55,530 --> 00:13:00,030 And this includes the numbering of the tRNA bases 194 00:13:00,030 --> 00:13:01,230 within that structure here. 195 00:13:03,840 --> 00:13:06,810 Just a point to make, this won't be a major focal point 196 00:13:06,810 --> 00:13:08,940 in the course, but do keep in mind 197 00:13:08,940 --> 00:13:12,480 that tRNA contains many post-transcriptionally modified 198 00:13:12,480 --> 00:13:16,560 bases, so you'll see an example of that in problem set one. 199 00:13:16,560 --> 00:13:19,890 Up to 25% of the bases can be modified. 200 00:13:19,890 --> 00:13:25,000 Typically, we see about 5% to 20% of them modified here. 201 00:13:25,000 --> 00:13:27,570 OK, you're not responsible for these structures, 202 00:13:27,570 --> 00:13:29,940 these modified structures, in the context of this class. 203 00:13:32,980 --> 00:13:36,460 So the key question for today is how 204 00:13:36,460 --> 00:13:42,870 are amino acids attached to the tRNA, as shown here? 205 00:13:42,870 --> 00:13:45,210 And in order for that to happen, there's 206 00:13:45,210 --> 00:13:50,240 a family of enzymes called aminoacyl-tRNA synthetases, 207 00:13:50,240 --> 00:13:53,330 or abbreviated aaRS. 208 00:13:53,330 --> 00:13:56,370 OK, so this name tells you right away, synthetase, 209 00:13:56,370 --> 00:14:00,090 that these enzymes use ATP. 210 00:14:00,090 --> 00:14:03,270 And these enzymes catalyze the attachment 211 00:14:03,270 --> 00:14:06,630 of amino acids to the three prime OH, 212 00:14:06,630 --> 00:14:13,230 or sometimes two prime OH, of the tRNA here for that. 213 00:14:13,230 --> 00:14:16,630 And so we're going to consider this overall reaction. 214 00:14:16,630 --> 00:14:18,630 And then we're going to think about the reaction 215 00:14:18,630 --> 00:14:22,950 mechanism and experiments that were done to give support 216 00:14:22,950 --> 00:14:27,090 to the mechanism that we see. 217 00:14:27,090 --> 00:14:30,030 So all aminoacyl-tRNA synthetases 218 00:14:30,030 --> 00:14:37,410 require ATP and hydrolyze ATP to AMP and PPI. 219 00:14:37,410 --> 00:14:40,140 And so they catalyze this overall reaction 220 00:14:40,140 --> 00:14:42,630 where we have an amino acid monomer. 221 00:14:42,630 --> 00:14:46,470 We have the tRNA that encodes this-- 222 00:14:46,470 --> 00:14:48,870 that is for this amino acid. 223 00:14:48,870 --> 00:14:56,220 ATP to give us the aminoacyl-tRNA AMP and PPI. 224 00:14:56,220 --> 00:15:00,740 So if the ATP is being hydrolyzed to AMP and PPI, 225 00:15:00,740 --> 00:15:02,730 what phosphate is being attacked? 226 00:15:02,730 --> 00:15:05,710 So we saw on Friday there's the alpha, beta, 227 00:15:05,710 --> 00:15:07,050 and gamma phosphates of ATP. 228 00:15:23,480 --> 00:15:25,740 OK, pardon? 229 00:15:25,740 --> 00:15:26,420 AUDIENCE: Beta. 230 00:15:26,420 --> 00:15:27,770 ELIZABETH NOLAN: Beta. 231 00:15:27,770 --> 00:15:29,042 Any takers? 232 00:15:29,042 --> 00:15:30,770 AUDIENCE: Alpha? 233 00:15:30,770 --> 00:15:32,820 ELIZABETH NOLAN: Any takers? 234 00:15:32,820 --> 00:15:33,320 Gamma? 235 00:15:36,790 --> 00:15:38,550 Yeah, so it's alpha. 236 00:15:38,550 --> 00:15:43,590 If you're getting AMP, it's attack at alpha. 237 00:15:43,590 --> 00:15:48,360 If you're getting ADP, it's attack at gamma here. 238 00:15:57,260 --> 00:16:01,860 OK, so P alpha is next door to the ribose of the nuc-- 239 00:16:01,860 --> 00:16:02,590 there. 240 00:16:02,590 --> 00:16:03,880 Yeah. 241 00:16:03,880 --> 00:16:06,470 OK. 242 00:16:06,470 --> 00:16:15,230 So if we consider this overall reaction, how does it work? 243 00:16:15,230 --> 00:16:18,710 Just before that, one other observation 244 00:16:18,710 --> 00:16:21,050 I just want to point out, if we're 245 00:16:21,050 --> 00:16:25,190 thinking about these enzymes and asking 246 00:16:25,190 --> 00:16:28,250 what is it that they recognize of the tRNA, 247 00:16:28,250 --> 00:16:30,140 so we have the anticodon. 248 00:16:30,140 --> 00:16:33,020 And that goes in hand-in-hand with the identity 249 00:16:33,020 --> 00:16:34,640 of the amino acid. 250 00:16:34,640 --> 00:16:37,370 Just keep in mind that it's not just the anticodon. 251 00:16:37,370 --> 00:16:41,930 So here we're seeing an example of an aminoacyl-tRNA synthetase 252 00:16:41,930 --> 00:16:44,240 with its tRNA bound. 253 00:16:44,240 --> 00:16:47,420 And we see that there's many contacts between the tRNA 254 00:16:47,420 --> 00:16:49,250 and this enzyme here. 255 00:16:49,250 --> 00:16:52,730 OK, so here we have the amino acid end, the anti-codon end, 256 00:16:52,730 --> 00:16:53,750 and all throughout here. 257 00:16:59,460 --> 00:17:05,220 So what is the mechanism to get us where we need to go? 258 00:17:05,220 --> 00:17:09,170 We have our overall reaction that I'll put up on the board, 259 00:17:09,170 --> 00:17:12,270 just to keep it straight as we move forward. 260 00:17:12,270 --> 00:17:25,529 So amino acid plus ATP plus the tRNA for that amino acid. 261 00:17:29,840 --> 00:17:40,430 Aminoacyl-tRNA synthetase to give us the aminoacyl-tRNA 262 00:17:40,430 --> 00:17:44,495 plus AMP plus PPI. 263 00:17:47,280 --> 00:17:48,990 So let's consider a mechanism. 264 00:17:55,350 --> 00:17:57,510 This is going to be a two-step mechanism. 265 00:18:00,800 --> 00:18:03,030 And so in the first step of this mechanism, 266 00:18:03,030 --> 00:18:20,180 we have the amino acid plus ATP. 267 00:18:24,940 --> 00:18:31,585 And we have formation of an OAMP intermediate. 268 00:18:37,760 --> 00:18:40,940 Plus PPI here. 269 00:18:40,940 --> 00:18:50,091 So this intermediate is called an amino adenylate. 270 00:18:55,650 --> 00:18:57,480 Adenlyate because adenosine here. 271 00:19:00,820 --> 00:19:04,260 And we need to think about why this intermediate might form. 272 00:19:04,260 --> 00:19:07,200 Why would we propose this in a mechanism? 273 00:19:07,200 --> 00:19:09,850 And then in step two-- 274 00:19:09,850 --> 00:19:12,540 we'll come back to that in a minute-- 275 00:19:12,540 --> 00:19:31,050 we can take our amino adenlyate, have our tRNA, 276 00:19:31,050 --> 00:19:35,700 this is the three prime end here. 277 00:19:35,700 --> 00:19:55,460 We can have attack with release of AMP. 278 00:19:55,460 --> 00:20:04,390 OK, so here we have the ester linkage at the three prime end, 279 00:20:04,390 --> 00:20:06,960 like what we see on that board here, 280 00:20:06,960 --> 00:20:11,060 to give us our aminoacyl-tRNA. 281 00:20:11,060 --> 00:20:13,330 OK, so we see in step one, there's 282 00:20:13,330 --> 00:20:16,840 formation of this amino adenylade intermediate. 283 00:20:16,840 --> 00:20:19,330 And in step two, there's transfer 284 00:20:19,330 --> 00:20:23,230 of the amino acid monomer to the three prime end of the tRNA 285 00:20:23,230 --> 00:20:25,330 here. 286 00:20:25,330 --> 00:20:32,780 So why might these enzymes go through that OAMP intermediate? 287 00:20:32,780 --> 00:20:35,190 What needs to happen for this chemistry to occur? 288 00:20:45,208 --> 00:20:47,250 AUDIENCE: You need a more activated reading group 289 00:20:47,250 --> 00:20:50,040 to have that acyl substitution form an ester 290 00:20:50,040 --> 00:20:51,292 from a carboxylate. 291 00:20:51,292 --> 00:20:52,250 ELIZABETH NOLAN: Right. 292 00:20:52,250 --> 00:20:56,100 We need to activate the CO2H group there. 293 00:20:56,100 --> 00:20:58,830 So this affords that. 294 00:20:58,830 --> 00:21:02,140 So what might be another possible mechanism, right? 295 00:21:02,140 --> 00:21:03,930 Imagine you're the experimentalist 296 00:21:03,930 --> 00:21:07,860 and you've combined your eighth amino acid ATP 297 00:21:07,860 --> 00:21:12,990 tRNA and this enzyme you've isolated in a test tube. 298 00:21:12,990 --> 00:21:16,270 And you see you've got this as a product. 299 00:21:16,270 --> 00:21:18,000 And this as a product. 300 00:21:18,000 --> 00:21:21,660 And you're wondering how did we get from reactants to products? 301 00:21:21,660 --> 00:21:23,460 This is one possibility. 302 00:21:23,460 --> 00:21:26,790 Maybe there's also a possibility of a concerted mechanism 303 00:21:26,790 --> 00:21:29,760 where there's no intermediate like the one I'm showing you 304 00:21:29,760 --> 00:21:30,660 here. 305 00:21:30,660 --> 00:21:32,220 These are just things to keep in mind 306 00:21:32,220 --> 00:21:34,680 when thinking about reactions. 307 00:21:34,680 --> 00:21:37,770 This two-step mechanism is the accepted mechanism 308 00:21:37,770 --> 00:21:40,440 for the amino aceyl tRNA synthetases. 309 00:21:40,440 --> 00:21:42,090 And so what we're going to think about 310 00:21:42,090 --> 00:21:45,120 are what are the experiments that were done 311 00:21:45,120 --> 00:21:49,170 to support this mechanism here. 312 00:21:49,170 --> 00:21:51,210 So what are the things we need to think about? 313 00:21:51,210 --> 00:21:52,793 And so we're going to think about this 314 00:21:52,793 --> 00:21:57,900 by examining one aminoacyl-tRNA synthetase as a paradigm. 315 00:21:57,900 --> 00:22:01,368 And this is the one for a isoleucine here. 316 00:22:28,180 --> 00:22:30,380 OK, so what are the experiments that 317 00:22:30,380 --> 00:22:33,290 need to be done to characterize this reaction 318 00:22:33,290 --> 00:22:34,640 and determine mechanism? 319 00:22:58,100 --> 00:22:59,190 OK. 320 00:22:59,190 --> 00:23:04,800 So one thing we need to confirm is reaction stoichiometry. 321 00:23:04,800 --> 00:23:08,460 So there's a stoichiometry up in what I've written above. 322 00:23:08,460 --> 00:23:12,250 But experimentally, that needs to be determined. 323 00:23:12,250 --> 00:23:21,360 So one, reaction stoichiometry. 324 00:23:21,360 --> 00:23:23,720 And so how can we think about this? 325 00:23:23,720 --> 00:23:27,020 We can think about the equivalence of the amino acid. 326 00:23:27,020 --> 00:23:28,900 So in this case, isoleucine. 327 00:23:28,900 --> 00:23:30,800 How many equivalents of isoleucine? 328 00:23:44,330 --> 00:23:47,410 And presumably, this isoleucine binds to the enzymes. 329 00:23:47,410 --> 00:23:48,910 We can think about it of equivalence 330 00:23:48,910 --> 00:23:50,620 of isoleucine bound. 331 00:23:50,620 --> 00:23:53,380 And we also see that ATP is consumed, right? 332 00:23:53,380 --> 00:23:55,690 That's hydrolyzed to AMP and PPI. 333 00:23:55,690 --> 00:24:00,100 So how many equivalents of ATP are consumed in this reaction? 334 00:24:13,990 --> 00:24:16,000 What else do we want to know? 335 00:24:16,000 --> 00:24:18,880 We need to know something about kinetics. 336 00:24:18,880 --> 00:24:21,850 So what are rates of formation? 337 00:24:21,850 --> 00:24:24,820 What is the rate of formation of the product, 338 00:24:24,820 --> 00:24:28,120 the aminoacyl-tRNA, and since I've 339 00:24:28,120 --> 00:24:30,250 told you this intermediate forms, 340 00:24:30,250 --> 00:24:33,736 what is the rate of formation of the intermediate? 341 00:24:33,736 --> 00:24:35,440 And since this is an intermediate, 342 00:24:35,440 --> 00:24:36,690 it's something transient. 343 00:24:36,690 --> 00:24:40,060 So we need to think about how are we as experimentalists 344 00:24:40,060 --> 00:24:42,370 going to detect this intermediate over the course 345 00:24:42,370 --> 00:24:44,590 of this reaction. 346 00:24:44,590 --> 00:24:48,280 It forms and decays in order to get product here. 347 00:24:52,850 --> 00:24:54,320 So rates of formation. 348 00:25:00,590 --> 00:25:14,335 And so we have formation of our product, which in this case-- 349 00:25:21,580 --> 00:25:35,292 and then formation of the intermediate, which 350 00:25:35,292 --> 00:25:36,500 I'll just abbreviate Ile-AMP. 351 00:25:40,930 --> 00:25:42,590 And what else would we like to know? 352 00:25:45,290 --> 00:25:47,720 We can figure out how, in addition 353 00:25:47,720 --> 00:25:52,490 to rate of formation of the product and the intermediate, 354 00:25:52,490 --> 00:26:06,430 we can think about the rate of transfer of Ile 355 00:26:06,430 --> 00:26:09,025 from the intermediate to the tRNA. 356 00:26:13,000 --> 00:26:16,330 So what this tells us is that we need 357 00:26:16,330 --> 00:26:26,965 a way to look for or detect the intermediate. 358 00:26:33,740 --> 00:26:34,240 Here. 359 00:26:39,100 --> 00:26:43,930 So imagine let's just have a hypothetical situation. 360 00:26:43,930 --> 00:26:47,110 If we find the intermediate, that tells us 361 00:26:47,110 --> 00:26:49,330 something about the reaction. 362 00:26:49,330 --> 00:26:51,005 If we don't find the intermediate, 363 00:26:51,005 --> 00:26:51,880 what can we conclude? 364 00:26:55,998 --> 00:26:56,960 Pardon? 365 00:26:56,960 --> 00:27:00,288 AUDIENCE: That there was no intermediate? 366 00:27:00,288 --> 00:27:02,080 ELIZABETH NOLAN: So that's one possibility. 367 00:27:02,080 --> 00:27:04,570 Are there other possibilities if our method doesn't 368 00:27:04,570 --> 00:27:07,840 let us detect the intermediate? 369 00:27:07,840 --> 00:27:09,989 AUDIENCE: Second step is to test. 370 00:27:13,250 --> 00:27:16,790 ELIZABETH NOLAN: Can it be hard to detect an intermediate? 371 00:27:16,790 --> 00:27:19,250 It can be very hard, right? 372 00:27:19,250 --> 00:27:21,830 So they don't always-- 373 00:27:21,830 --> 00:27:23,690 there aren't around all the time very much 374 00:27:23,690 --> 00:27:26,240 or in very abundant quantities. 375 00:27:26,240 --> 00:27:30,650 So if it's not detected, could it be there? 376 00:27:30,650 --> 00:27:31,850 Yeah, it might be there. 377 00:27:31,850 --> 00:27:35,730 And the method just didn't allow for it to be seen. 378 00:27:35,730 --> 00:27:38,743 So you always need to keep that possibility in mind. 379 00:27:38,743 --> 00:27:40,160 This will be a case where there is 380 00:27:40,160 --> 00:27:42,800 a robust method that allows us to detect 381 00:27:42,800 --> 00:27:44,980 this type of intermediate. 382 00:27:44,980 --> 00:27:47,420 But always keep that in mind. 383 00:27:47,420 --> 00:27:52,880 OK, so first thinking about reaction stoichiometry. 384 00:27:52,880 --> 00:27:55,340 We're not going to go over the experiments that 385 00:27:55,340 --> 00:27:56,930 were done to define this. 386 00:27:56,930 --> 00:27:59,450 I'll just tell you some facts that result 387 00:27:59,450 --> 00:28:01,890 from some experimental studies. 388 00:28:01,890 --> 00:28:06,740 So this isoleucine aminoacyl-tRNA synthetase 389 00:28:06,740 --> 00:28:09,560 binds 1 equivalent of isoleucine as indicated 390 00:28:09,560 --> 00:28:11,750 in the overall reaction. 391 00:28:11,750 --> 00:28:15,380 And it consumes one equivalent of ATP, 392 00:28:15,380 --> 00:28:17,870 also as shown in this overall reaction, 393 00:28:17,870 --> 00:28:21,860 to make one equivalent of the aminoacyl-tRNA. 394 00:28:21,860 --> 00:28:26,760 OK and these stoichiometries were determined experimentally. 395 00:28:26,760 --> 00:28:31,010 So now we need to think about points two and three 396 00:28:31,010 --> 00:28:33,840 to characterize the reaction kinetics. 397 00:28:33,840 --> 00:28:36,242 So what experiments were done? 398 00:28:36,242 --> 00:28:37,700 So there are several different sets 399 00:28:37,700 --> 00:28:40,040 of experiments, some of which we're familiar 400 00:28:40,040 --> 00:28:44,120 with from 7.05 or 5.07 and others that will be new 401 00:28:44,120 --> 00:28:46,700 and presented in more detail in recitation this week 402 00:28:46,700 --> 00:28:48,090 and next week. 403 00:28:48,090 --> 00:28:52,580 So we can imagine doing steady state kinetic experiments, 404 00:28:52,580 --> 00:28:55,880 as well as pre-steady state kinetic experiments. 405 00:28:55,880 --> 00:28:58,280 And the general aims here are, one, 406 00:28:58,280 --> 00:29:02,450 to determine the rate of aminoacyl-tRNA formation, 407 00:29:02,450 --> 00:29:05,480 to determine the rate of amino adenylate formation, 408 00:29:05,480 --> 00:29:07,160 so this intermediate-- and again, we 409 00:29:07,160 --> 00:29:09,800 need a method to detect the intermediate. 410 00:29:09,800 --> 00:29:11,300 And at the end of the day, we'd like 411 00:29:11,300 --> 00:29:15,380 to know what is the rate determining step. 412 00:29:15,380 --> 00:29:18,470 So a method that is commonly employed 413 00:29:18,470 --> 00:29:20,360 for these types of studies involves 414 00:29:20,360 --> 00:29:22,460 the use of radioactivity. 415 00:29:22,460 --> 00:29:27,020 And we'll just go over a few points about radioactivity now 416 00:29:27,020 --> 00:29:28,940 to help with understanding these experiments. 417 00:29:28,940 --> 00:29:33,110 And you'll hear more about this method in recitation this week. 418 00:29:33,110 --> 00:29:35,750 So the experiments I'm going to tell you about 419 00:29:35,750 --> 00:29:42,800 are going to involve the use of radio isotopes like C14, P32. 420 00:29:42,800 --> 00:29:45,140 And the question is, why do we like 421 00:29:45,140 --> 00:29:48,980 to use radio isotopes in biochemical experiments? 422 00:29:48,980 --> 00:29:51,170 And they're really excellent probes. 423 00:29:51,170 --> 00:29:53,000 It's the bottom line. 424 00:29:53,000 --> 00:29:56,420 And one reason for that is that if you can use a radio isotope 425 00:29:56,420 --> 00:30:00,200 like C14 or P32, it's introducing 426 00:30:00,200 --> 00:30:04,250 minimal perturbation into your system. 427 00:30:04,250 --> 00:30:07,490 So you're not needing to attach a fluorophore whether it be 428 00:30:07,490 --> 00:30:09,410 a small molecule or a protein. 429 00:30:09,410 --> 00:30:12,050 You're not modifying the structure 430 00:30:12,050 --> 00:30:14,910 of a component of your system. 431 00:30:14,910 --> 00:30:17,600 So the overall size and the chemical properties 432 00:30:17,600 --> 00:30:20,540 are maintained when you use different isotopes 433 00:30:20,540 --> 00:30:22,150 of the same element. 434 00:30:22,150 --> 00:30:24,510 And some of the ones we'll see today 435 00:30:24,510 --> 00:30:30,200 are, for instance, C14 labeled isoleucine, P32 labeled ATP. 436 00:30:30,200 --> 00:30:31,940 They have the same chemical properties 437 00:30:31,940 --> 00:30:34,850 as the unlabeled forms, and same size. 438 00:30:34,850 --> 00:30:36,800 The other point to make is that we 439 00:30:36,800 --> 00:30:40,590 can detect very small amounts of radioactivity in a sample. 440 00:30:40,590 --> 00:30:43,520 And you'll see some of those calculations 441 00:30:43,520 --> 00:30:46,490 and how to do them in recitation this week. 442 00:30:46,490 --> 00:30:49,010 So we can detect small amounts, and that's 443 00:30:49,010 --> 00:30:51,860 good for looking for something like an intermediate. 444 00:30:51,860 --> 00:30:55,910 And there's readily available techniques 445 00:30:55,910 --> 00:31:00,260 for quantifying radioactivity in a sample. 446 00:31:00,260 --> 00:31:02,450 So if you see nomenclature like this, 447 00:31:02,450 --> 00:31:06,110 the NX nomenclature indicates the radioisotope 448 00:31:06,110 --> 00:31:08,390 in this sample. 449 00:31:08,390 --> 00:31:11,860 And I'll just say in passing here, 450 00:31:11,860 --> 00:31:13,910 we all know the isotopes are atoms 451 00:31:13,910 --> 00:31:16,760 bearing the same number of protons but different numbers 452 00:31:16,760 --> 00:31:18,710 of neutrons. 453 00:31:18,710 --> 00:31:21,530 And radioactive isotopes have an unstable nucleus, 454 00:31:21,530 --> 00:31:24,740 which means there's a radioactive decay. 455 00:31:24,740 --> 00:31:29,140 And typically-- well, we often use beta emitters 456 00:31:29,140 --> 00:31:30,470 in biochemical studies. 457 00:31:30,470 --> 00:31:33,380 And that's what you'll see today. 458 00:31:33,380 --> 00:31:36,190 So what are some of the experiments? 459 00:31:38,720 --> 00:31:42,170 We're first going to consider looking at the steady state 460 00:31:42,170 --> 00:31:48,140 kinetics to ask what do we learn in the steady state. 461 00:31:56,970 --> 00:32:06,780 So from our steady state experiments, 462 00:32:06,780 --> 00:32:11,580 we're able to get our Kcat and our Km 463 00:32:11,580 --> 00:32:15,630 and the catalytic efficiency, which is the Kcat over Km. 464 00:32:15,630 --> 00:32:21,630 We're going to compare our Kcat values or turnover today. 465 00:32:21,630 --> 00:32:28,875 So experiment one is to monitor formation of product. 466 00:32:39,890 --> 00:32:41,690 So how is this done? 467 00:32:41,690 --> 00:32:45,110 This reaction is done by taking C14 labeled 468 00:32:45,110 --> 00:32:49,640 isoleucine and unlabeled tRNA and watching 469 00:32:49,640 --> 00:32:52,550 for transfer of that radio label to the tRNA. 470 00:33:14,550 --> 00:33:18,290 And so what comes from these studies 471 00:33:18,290 --> 00:33:23,410 is a Kcat on the order of 1.4 per second. 472 00:33:30,750 --> 00:33:35,970 And now we have a way to detect this amino adenylate 473 00:33:35,970 --> 00:33:36,930 intermediate. 474 00:33:36,930 --> 00:33:38,970 And we'll talk about that assay in a minute, 475 00:33:38,970 --> 00:33:41,370 after we get through this comparison. 476 00:33:41,370 --> 00:33:45,630 We do a steady state experiment to monitor 477 00:33:45,630 --> 00:33:52,652 formation of this amino adenylate intermediate. 478 00:33:55,200 --> 00:33:58,650 And this assay also uses radioactivity. 479 00:33:58,650 --> 00:34:04,425 And it's called ATP PPI exchange assay. 480 00:34:04,425 --> 00:34:06,300 And we'll go over how this works in a minute. 481 00:34:09,130 --> 00:34:11,760 So the results of these experiments 482 00:34:11,760 --> 00:34:17,909 give a Kcat on the order of 80 per second. 483 00:34:23,100 --> 00:34:25,790 So what does this comparison tell you? 484 00:34:36,550 --> 00:34:40,300 These values are quite different, correct? 485 00:34:40,300 --> 00:34:47,949 So we're seeing that this ATP PPI exchange assay 486 00:34:47,949 --> 00:34:51,489 is telling us that ATP PPI exchange, which 487 00:34:51,489 --> 00:34:54,130 is a measure of formation of this intermediate, 488 00:34:54,130 --> 00:35:00,010 is about 60-fold faster than formation of product here. 489 00:35:00,010 --> 00:35:03,910 That's an important observation to have. 490 00:35:03,910 --> 00:35:07,090 So how are we going to figure this out? 491 00:35:07,090 --> 00:35:10,180 How are we going to see this intermediate? 492 00:35:10,180 --> 00:35:13,750 That's the question we need to ask next. 493 00:35:13,750 --> 00:35:22,960 And so we need to go over this ATP PPI exchange assay. 494 00:35:22,960 --> 00:35:29,140 And this is an assay that will come up again in module 4 495 00:35:29,140 --> 00:35:32,230 when we talk about the biosynthesis 496 00:35:32,230 --> 00:35:33,770 of non-ribosomal peptides. 497 00:35:33,770 --> 00:35:36,835 So we'll return to this type of assay and data many times. 498 00:35:40,210 --> 00:36:02,080 So the question is, if we have this reaction, OK, 499 00:36:02,080 --> 00:36:03,400 how do we detect this? 500 00:36:08,590 --> 00:36:10,200 OK, it's not so easy. 501 00:36:10,200 --> 00:36:12,170 And we need an assay. 502 00:36:12,170 --> 00:36:14,150 And this is some of the background 503 00:36:14,150 --> 00:36:17,600 towards the development of this assay. 504 00:36:17,600 --> 00:36:23,720 So we need to suppose that our amino acid and ATP react 505 00:36:23,720 --> 00:36:26,330 with the aminoacyl-tRNA synthetase 506 00:36:26,330 --> 00:36:29,110 in the absence of tRNA. 507 00:36:29,110 --> 00:36:32,660 And that's indicated by step one, more or less. 508 00:36:32,660 --> 00:36:35,280 But that doesn't show it experimentally. 509 00:36:35,280 --> 00:36:38,540 So in the absence of tRNA, this amino acid and ATP 510 00:36:38,540 --> 00:36:42,770 react with the enzyme and they form the aminoacyl AMP 511 00:36:42,770 --> 00:36:45,260 intermediate and PPI. 512 00:36:45,260 --> 00:36:47,570 And they do this reversibly. 513 00:36:47,570 --> 00:36:50,480 OK, so the reversibility of this reaction 514 00:36:50,480 --> 00:36:54,380 is key for ATP PPI exchange to work. 515 00:36:57,430 --> 00:37:03,220 So if this occurs and they do this reversibly, 516 00:37:03,220 --> 00:37:09,310 therefore we can deduce formation of the aminoacyl AMP. 517 00:37:09,310 --> 00:37:14,320 If we add radio labeled PPI, the amino acid, and ATP 518 00:37:14,320 --> 00:37:18,670 to the enzyme and we see that radio labeled phosphorus 519 00:37:18,670 --> 00:37:24,010 from the radio labeled PPI incorporate into ATP. 520 00:37:24,010 --> 00:37:28,150 That's only going to happen if this chemistry is reversible. 521 00:37:28,150 --> 00:37:31,210 And bear in mind, we can detect very small quantities 522 00:37:31,210 --> 00:37:34,240 with radioactivity. 523 00:37:34,240 --> 00:37:36,520 So it's not that it has to be reversible 524 00:37:36,520 --> 00:37:38,350 to some large degree. 525 00:37:38,350 --> 00:37:42,550 We're relying on the detection of this radio label. 526 00:37:42,550 --> 00:37:45,700 So how does this work chemically? 527 00:37:45,700 --> 00:37:46,580 Let's take a look. 528 00:37:50,220 --> 00:37:54,930 OK, so imagine here we have our ATP. 529 00:37:54,930 --> 00:37:58,022 We have our amino acid. 530 00:37:58,022 --> 00:37:58,980 And we have our enzyme. 531 00:38:02,430 --> 00:38:04,275 And step one, we have binding. 532 00:38:09,300 --> 00:38:15,390 So there's some ATP binding site to the enzyme and some site 533 00:38:15,390 --> 00:38:16,800 for the amino acid to bind. 534 00:38:16,800 --> 00:38:19,470 And I'm leaving magnesium out of this depiction, 535 00:38:19,470 --> 00:38:22,950 but remember that magnesium and ATP come together. 536 00:38:22,950 --> 00:38:23,490 Now what? 537 00:38:23,490 --> 00:38:28,020 Step two, OK, we're going to have 538 00:38:28,020 --> 00:38:41,280 a chemical step where we have formation 539 00:38:41,280 --> 00:38:47,730 of the amino adenylate and PPI. 540 00:38:47,730 --> 00:38:50,295 And they currently are bound to the enzyme. 541 00:38:53,010 --> 00:38:53,975 We have step three. 542 00:38:59,130 --> 00:39:05,620 So imagine in this step our PPI is released. 543 00:39:12,600 --> 00:39:15,960 And this is another key aspect of this assay. 544 00:39:15,960 --> 00:39:17,520 So what does this mean? 545 00:39:17,520 --> 00:39:20,550 We now need to think about going backwards. 546 00:39:20,550 --> 00:39:25,620 If the PPI is released and we spike this reaction with radio 547 00:39:25,620 --> 00:39:28,980 labeled PPI and work our way backwards, 548 00:39:28,980 --> 00:39:32,660 will the radio label end up here in the ATP? 549 00:39:32,660 --> 00:39:33,660 OK. 550 00:39:33,660 --> 00:39:40,450 So this is going to be going backwards. 551 00:39:40,450 --> 00:39:47,260 We've left off with this enzyme with the amino adenylate bound. 552 00:39:47,260 --> 00:39:51,310 We have the PPI that was released. 553 00:39:51,310 --> 00:39:57,280 And then we spike this reaction with our radio labeled 554 00:39:57,280 --> 00:39:58,800 or hot PPI. 555 00:40:10,260 --> 00:40:12,000 So then what happens? 556 00:40:12,000 --> 00:40:15,480 Step four, working backwards. 557 00:40:21,610 --> 00:40:26,245 Imagine that some of the radio labeled PPI binds. 558 00:40:32,300 --> 00:40:34,340 Then what? 559 00:40:34,340 --> 00:40:35,930 Working backwards another step. 560 00:40:41,690 --> 00:40:45,075 32 P ATP and the amino acid. 561 00:40:51,910 --> 00:41:00,590 And then we have release here. 562 00:41:00,590 --> 00:41:04,180 OK, so then the question is, can you detect this? 563 00:41:12,250 --> 00:41:16,320 And so if you can detect some incorporation of this radio 564 00:41:16,320 --> 00:41:19,590 label into the ATP, that indicates 565 00:41:19,590 --> 00:41:23,163 that this enzyme worked through that type of intermediate. 566 00:41:26,544 --> 00:41:28,790 AUDIENCE: So are PPI not also sometimes 567 00:41:28,790 --> 00:41:34,340 [INAUDIBLE] and then if you had some competing hypothesis where 568 00:41:34,340 --> 00:41:39,240 it made ATP and ADP, then your PPI would maybe sometimes 569 00:41:39,240 --> 00:41:43,524 turn into just a single radio label 570 00:41:43,524 --> 00:41:46,350 phosphate that could then have the same reverse reactions 571 00:41:46,350 --> 00:41:47,823 as the [INAUDIBLE]? 572 00:41:47,823 --> 00:41:48,740 ELIZABETH NOLAN: Yeah. 573 00:41:48,740 --> 00:41:53,660 So whether you initially end up with PPI or PI 574 00:41:53,660 --> 00:41:57,770 is going to depend on how the ATP is hydrolyzed. 575 00:41:57,770 --> 00:42:01,250 And so you could imagine maybe there 576 00:42:01,250 --> 00:42:04,200 could be some background ATP hydrolysis 577 00:42:04,200 --> 00:42:07,700 that gives ADP and PI in this type of assay. 578 00:42:07,700 --> 00:42:11,492 That's something you always need to look out for. 579 00:42:11,492 --> 00:42:14,090 For the purpose of this, let's assume 580 00:42:14,090 --> 00:42:18,020 that we're not having some background problem in terms 581 00:42:18,020 --> 00:42:24,710 of the ATP source, and also that the enzyme is specific in terms 582 00:42:24,710 --> 00:42:28,130 of what it's doing to the ATP. 583 00:42:28,130 --> 00:42:31,670 But yeah, certainly background ATP hydrolysis 584 00:42:31,670 --> 00:42:34,110 can be a problem. 585 00:42:34,110 --> 00:42:39,560 So how will this be detected? 586 00:42:46,290 --> 00:42:51,550 And how will you know the radio label is associated with ATP 587 00:42:51,550 --> 00:42:53,175 and not something else in your mixture? 588 00:42:58,210 --> 00:42:59,160 AUDIENCE: [INAUDIBLE] 589 00:42:59,160 --> 00:43:00,700 ELIZABETH NOLAN: Pardon? 590 00:43:00,700 --> 00:43:01,627 AUDIENCE: [INAUDIBLE] 591 00:43:01,627 --> 00:43:02,460 ELIZABETH NOLAN: No. 592 00:43:02,460 --> 00:43:05,190 So we're going to look at the radioactivity. 593 00:43:05,190 --> 00:43:08,460 So this will come up more in recitation this week. 594 00:43:08,460 --> 00:43:11,850 But we need to be able to measure radioactivity by, say, 595 00:43:11,850 --> 00:43:14,640 scintillation counting here. 596 00:43:14,640 --> 00:43:16,710 But what's also needed is a separation 597 00:43:16,710 --> 00:43:19,800 because you need to know where that signal's coming from. 598 00:43:19,800 --> 00:43:22,710 You need to know it's coming from ATP 599 00:43:22,710 --> 00:43:26,070 and, say, not a background from however much of the PPI 600 00:43:26,070 --> 00:43:28,470 you introduced. 601 00:43:28,470 --> 00:43:31,590 Or if you have no idea what's going on with your chemistry, 602 00:43:31,590 --> 00:43:35,530 maybe the data are going to tell you it's not this mechanism. 603 00:43:35,530 --> 00:43:38,340 So you need to have a separation. 604 00:43:38,340 --> 00:43:41,910 So how might you separate ATP from all 605 00:43:41,910 --> 00:43:43,260 of these other components? 606 00:43:48,561 --> 00:43:50,020 AUDIENCE: Based on affinity column. 607 00:43:50,020 --> 00:43:51,900 ELIZABETH NOLAN: Some affinity column. 608 00:43:51,900 --> 00:43:54,150 So I like the column. 609 00:43:54,150 --> 00:43:57,810 But we're not going to have some sort of tag on the ATP. 610 00:43:57,810 --> 00:43:59,720 That might be a problem for that enzyme. 611 00:43:59,720 --> 00:44:01,830 But your notion is correct in the sense 612 00:44:01,830 --> 00:44:04,350 that we'll use some sort of chromatography 613 00:44:04,350 --> 00:44:06,230 in order to separate. 614 00:44:06,230 --> 00:44:08,700 OK, so maybe HPLC, how many of you 615 00:44:08,700 --> 00:44:12,613 have used an HPLC or at least know what one is? 616 00:44:12,613 --> 00:44:13,530 AUDIENCE: [INAUDIBLE]. 617 00:44:13,530 --> 00:44:14,488 ELIZABETH NOLAN: Right. 618 00:44:14,488 --> 00:44:17,640 So typically looking at UV vis. 619 00:44:17,640 --> 00:44:21,420 But you can imagine hooking up an HPLC to a detector that 620 00:44:21,420 --> 00:44:24,930 allows you to do scintillation counting and some sort 621 00:44:24,930 --> 00:44:29,760 of column that will allow you to look for ATP. 622 00:44:29,760 --> 00:44:35,660 Is all of the ATP going to be radioactive in this assay? 623 00:44:35,660 --> 00:44:37,560 No. 624 00:44:37,560 --> 00:44:40,740 So again, we can detect small quantities. 625 00:44:40,740 --> 00:44:43,260 And as long as there's a little bit of reversibility, 626 00:44:43,260 --> 00:44:46,380 we can see this here. 627 00:44:46,380 --> 00:44:50,190 OK, so what's critical in this assay 628 00:44:50,190 --> 00:44:53,160 is the reversibility of steps 3 and 4. 629 00:44:57,140 --> 00:45:00,170 What would happen in this assay if the PPI is not released? 630 00:45:06,783 --> 00:45:08,710 AUDIENCE: [INAUDIBLE]. 631 00:45:08,710 --> 00:45:09,828 ELIZABETH NOLAN: Right. 632 00:45:09,828 --> 00:45:11,620 Under the conditions, or if for some reason 633 00:45:11,620 --> 00:45:14,500 the PPI is not released, we're not 634 00:45:14,500 --> 00:45:17,350 going to see this exchange reaction. 635 00:45:17,350 --> 00:45:20,290 We're going to have a readout that doesn't give us this. 636 00:45:20,290 --> 00:45:21,700 Does that mean this didn't form? 637 00:45:24,620 --> 00:45:26,030 No. 638 00:45:26,030 --> 00:45:28,920 OK, so there's many caveats and details 639 00:45:28,920 --> 00:45:32,100 that you need to think through when thinking about a reaction 640 00:45:32,100 --> 00:45:36,220 and then the experiment is done to test this. 641 00:45:36,220 --> 00:45:40,620 So in the case of these aminoacyl-tRNA synthetases, 642 00:45:40,620 --> 00:45:44,970 these ATP PPI exchange assays work well. 643 00:45:44,970 --> 00:45:48,750 And these assays can be used to get steady state kinetic 644 00:45:48,750 --> 00:45:53,940 parameters, to get Kcat, Km, Kcat over Km, which 645 00:45:53,940 --> 00:45:58,890 is where this type of value comes from, in this case here. 646 00:46:05,490 --> 00:46:12,510 So back to these analyses up here, what they're telling us 647 00:46:12,510 --> 00:46:16,350 is that formation of this amino adenylate intermediate 648 00:46:16,350 --> 00:46:21,800 is about 60-fold faster than formation of the product. 649 00:46:21,800 --> 00:46:23,280 OK. 650 00:46:23,280 --> 00:46:25,110 And what we all want to recall when 651 00:46:25,110 --> 00:46:28,050 thinking about steady state experiments 652 00:46:28,050 --> 00:46:31,400 is that they're set up with a great excess of substrate 653 00:46:31,400 --> 00:46:33,960 and with the enzyme concentration. 654 00:46:33,960 --> 00:46:36,678 The reaction is zero order in respect to substrate. 655 00:46:36,678 --> 00:46:38,220 And you'll have some additional notes 656 00:46:38,220 --> 00:46:43,450 about that in your recitation materials this week for review. 657 00:46:43,450 --> 00:46:46,110 So something else biochemists like 658 00:46:46,110 --> 00:46:50,820 to do when looking at reactions and understanding reaction 659 00:46:50,820 --> 00:46:55,380 mechanisms is to look in the pre-steady state. 660 00:46:55,380 --> 00:46:57,870 And this came up briefly in lecture 1 as a method. 661 00:46:57,870 --> 00:46:59,370 And again, you'll hear more about it 662 00:46:59,370 --> 00:47:02,580 in recitation over the next two weeks. 663 00:47:02,580 --> 00:47:05,850 In these experiments, the goal is 664 00:47:05,850 --> 00:47:10,740 to look at the very first, early moments of a reaction. 665 00:47:10,740 --> 00:47:14,010 And they're set up quite differently. 666 00:47:14,010 --> 00:47:18,150 So limiting substrate is used. 667 00:47:18,150 --> 00:47:21,210 There's no turnover, so huge contrast 668 00:47:21,210 --> 00:47:24,570 to what we know about steady state experiments. 669 00:47:24,570 --> 00:47:27,870 And one of the goals is to look at the formation 670 00:47:27,870 --> 00:47:31,110 and consumption of intermediates here. 671 00:47:31,110 --> 00:47:36,080 So this type of chemistry often happens on a fast timescale. 672 00:47:36,080 --> 00:47:40,180 You can imagine millisecond timescale here, 673 00:47:40,180 --> 00:47:44,250 which means that we need a special apparatus that has fast 674 00:47:44,250 --> 00:47:46,920 mixing capabilities, because there's no way for one of us 675 00:47:46,920 --> 00:47:49,520 to do this on our own with our pipette. 676 00:47:52,350 --> 00:47:55,770 And so the type of experiment or apparatus 677 00:47:55,770 --> 00:47:58,020 used is called a stop flow. 678 00:47:58,020 --> 00:48:01,890 And I just show one depiction of a stop flow apparatus here. 679 00:48:01,890 --> 00:48:06,210 You'll get some other variations on this theme in the recitation 680 00:48:06,210 --> 00:48:07,530 notes. 681 00:48:07,530 --> 00:48:09,600 OK, but effectively what happens is 682 00:48:09,600 --> 00:48:14,120 that you have two drive syringes, a and b, 683 00:48:14,120 --> 00:48:17,700 and each of these syringes will contain certain components 684 00:48:17,700 --> 00:48:19,590 of your reaction. 685 00:48:19,590 --> 00:48:23,690 And this stop flow has a drive motor and a stop syringe. 686 00:48:23,690 --> 00:48:26,280 And it effectively allows you to rapidly mix 687 00:48:26,280 --> 00:48:31,860 the components of these syringes in a mixer, shown here. 688 00:48:31,860 --> 00:48:35,790 And then you either have some way to detect product-- 689 00:48:35,790 --> 00:48:38,430 so maybe if you can use optical absorption, 690 00:48:38,430 --> 00:48:43,470 you have a UV vis detector or a fluorescence detector. 691 00:48:43,470 --> 00:48:46,530 Or in other cases what you'll do is 692 00:48:46,530 --> 00:48:49,660 you'll punch the reaction at a certain time point. 693 00:48:49,660 --> 00:48:53,580 So you need a third syringe not shown here with a quencher. 694 00:48:53,580 --> 00:48:56,340 So you can imagine if you're working with an enzyme, 695 00:48:56,340 --> 00:49:00,120 maybe you quench by addition of acid or base, something 696 00:49:00,120 --> 00:49:03,390 that will denature and precipitate that enzyme. 697 00:49:03,390 --> 00:49:05,130 And then you can take that sample 698 00:49:05,130 --> 00:49:09,570 and analyze it in some way that fits in terms of what 699 00:49:09,570 --> 00:49:12,390 you need to detect there. 700 00:49:12,390 --> 00:49:17,190 So this type of methodology was used in order 701 00:49:17,190 --> 00:49:24,690 to monitor transfer of isoleucine to its tRNA. 702 00:49:24,690 --> 00:49:28,470 And so where we'll pick up in lecture on Wednesday 703 00:49:28,470 --> 00:49:33,210 is the design of that experiment in terms of what will we 704 00:49:33,210 --> 00:49:35,610 put in each syringe, and then what 705 00:49:35,610 --> 00:49:38,070 are the results of those experiments? 706 00:49:38,070 --> 00:49:42,900 And ultimately, what does that tell us about rates of transfer 707 00:49:42,900 --> 00:49:45,450 here? 708 00:49:45,450 --> 00:49:47,420 That's where we'll continue.