Is your mic work? No. OK. Yes. All right. We actually have a lot of announcements today. And I want my teaching time. So we're going to get started right away with the announcement phase of the class. So a few students emailed some announcements. I guess there's a performance coming up. You guys are so talented. It just blows me away. Look at this. You got the spice it up going on. You all should go to that. It's spiced up. And we also have this global brigades. So if you have any more questions, the email is at the bottom. We have a recruitment event coming. So this is becoming the announcement central. So we actually have a very special guest here today, Professor Nick Fawzi, who's in the pharmacology or MPP department. And he wanted to share with you some great news about a new class that he's starting. Thanks, Art. My name is Nick Fawzi. I'm a new professor here in biology. And I'm starting a class in the fall entitled Bimolecular Interactions, Health, Disease, and Drug Design. And the idea of the class, one of the prerequisites is this class that you're in right now. The class is directed towards mainly seniors, as well as a few graduate students. But juniors and seniors are welcome to apply. We're going to process the wait list in the fall. But the idea of the class is to understand more about the physical interactions that make life possible, what goes wrong in disease, and then how you can use design techniques, both experimental and computer, to solve some of these problems. So we're going to have computational aspects. We're going to have experimental aspects, guest lectures from industry, and as well as other faculty here at Brown. So it should be an exciting seminar class. Thanks very much. All right. So this is actually my favorite lecture of the semester. So I might go a little fast. Slow me down with questions. So here we're sort of bringing a lot of things together. And we're going to start talking about, well, first we have one of my reminders. Monty Python provides a little not so funny comic relief. So there's actually exams coming up. There's exam three. It's April 29th, Tuesday. Guests two Tuesdays from today. So something you want to begin thinking about. The lecture is covered on that 16th through 22. And the final, remember that's a comprehensive final. And that's coming up on Monday, May 12th. And we have a new location that has been awarded to us for the final. And so we're going to leave Sesame Street, Burt, and we're going to go to Met Kim. So Met Kim is for the N2Z folks. Met Kim is across the street. I guess Burt was already claimed. And so we're going to be in Met Kim and MacMillan for the rest of us. So those who are normally in MacMillan will stay in MacMillan for the final examination. Yes. Met Kim is across the street. Met Kef, yeah. Yeah, the registrar calls it Met Kim. I guess at one point it used to be associated with chemistry. And now it's Met Kim across the street, yeah. So if you Google Brown Met Kim, the first hit, that's where you need to go. All right, so let's move along. So today we're going to be covering translation. Remember, we covered transcription last time. We've made our mRNA molecules. And now we need to convert those RNA molecules into proteins. And so actually, one of the world leaders in research into translation is Professor Garwald Yogel. And so he actually solves crystal structures of ribosomes. And these are millions of Dalton's large complexes. And so we definitely have sort of a proud tradition at Brown of pioneering research in this field. And so first, we need to think about the genetic code. And so we have to think about, OK, we have these four nucleotides, and we've got 20 amino acids. So we've got to translate these two languages. And so we have to think about the codons, what's actually recognized by the tRNA molecules. And we also have to think about some of the diversity of different types of tRNA molecules and why things have evolved as they have. And then we're going to look, after considering the code at the actual translation machinery, we all know that it's messenger RNA, tRNAs, ribosomes coming together. And we're going to be going through this process in a series of steps. We typically see initiation, elongation, and termination. But activation of the amino acids charging of the tRNAs is important. And also, post-translational processing and folding and localizations of proteins is also important. One of the great tools to study the process of translation is antibiotics. And antibiotics often can interact with the ribosomal machinery itself, and so you can get some mechanistic insights into translation. So let me see if I can make my pointer not go away. So this is the actual translator for a sky-high view of the translator, so the tRNA molecule. And so you have a codon that's recognized in the messenger RNA. And there's a so-called anti-codon that base pairs in an anti-parallel fashion with this codon. The tRNA molecule obviously is a very large RNA molecule. And at the three prime end of this RNA molecule is the position that gets charged with the amino acid. And so through an ester linkage to the carboxylate of each of the 20 amino acids, we actually activate the tRNA for its role in synthesis of polypeptide. And so we're going to be looking at detail at this. But let's first think about the genetic code. So when you think about it, just from first principles, we've only got four nucleotides. So it couldn't possibly be a one-letter code. We have 20 amino acids, not four amino acids. And it really, it's hard to imagine how it could be a two-letter code, because four to the two is only 16. We need 20 different unique combinations to be able to specify all the naturally occurring amino acids. Now, three bases, that would get us in the ballpark. So that would have 64 possible combinations. But who knows? Nature could have evolved a four-letter code or a five-letter code. So three would probably be sufficient. It's hard to imagine why you would need more. But so that is one of the areas that was investigated in discovering the genetic code, how many nucleotides are involved in each binding to the tRNA. And then also, well, say if you do have a triplet code, then there's two other possibilities. Is that code going to be overlapping or non-overlapping? It's sort of hard to imagine, well, how could it be overlapping? If we're overlapping, you would have this nesting of the codons. And so you would put artificial limitations into the types of polypeptides that could be attached to each other. They would have to mesh with each other. So for example, here, the AUA that would bind in a code for addition of one amino acid, but then UAC, ACG. So that was a little bit. I mean, we had to discount that possibility. It seemed unlikely. The other possibility is the non-overlapping code, where you need to pick a so-called reading frame. And once you figure out exactly where to start, then you read off amino acid coding codons one at a time in sequence. And so one of the very early experiments that was done to figure out whether it was non-overlapping or overlapping was to make point mutations. And so if it were an overlapping code, if you made a point mutation, you would affect the incorporation of multiple amino acids potentially into the polypeptide. Whereas if we're non-overlapping, you would only change potentially the incorporation of a single amino acid. So that was figured out pretty quickly that it indeed was a non-overlapping code. But how did we figure out there was a triplet code? And so here's this concept of reading frame. If it is non-overlapping, where we start the interpretation vastly affects the type of polypeptide we synthesize. So if you look at each of these three reading frames, you have a completely different set of triplets here. And so we need to have some mechanism to precisely, at the single nucleotide level, figure out where to start reading this frame. And so here's another way to look at it. So if you look at it in the nucleotide letters, we don't speak nucleotides, but we speak English, at least most of us. And so this reading frame, so here we have a sequence of letters. It's all misched together. But if we do this particular reading frame, hey, I know what that means. A big red fox ate some eggs. Cool. But then if you go into these other reading frames, we depart from the land of English into Klingon.  That doesn't make any sense to me. And so this sort of in a silly way gets at the importance of picking the right frame. And only one of these frames will make sense in terms of the correct polypeptide being synthesized. And so these are the types of experiments that was in the 1960s or so. People were figuring this out. And so people did a series of different experiments. So they, for example, if you just do a one base substitution, they found, as I mentioned before, indeed it's not a non-over. It is a non-overlapping code because they just changed the incorporation of a single amino acid. You just change one of these codons. But say what happens if you delete a nucleotide? Well, that shifts the remaining nucleotides to the left. Completely throwing the interpretation out of frame. So we have English followed by Klingon. So what if we insert? Same sort of thing happens. We throw it out of frame. We get a completely different set of triplets. OK, what if we insert and then delete? Well, straddling those two modifications is English. And so that was helpful to know that we could regain frame. It's probably obvious. But then these two experiments really got at whether it's a triplet code or a four letter code, five letter code. So they did two base insertions, threw it out of frame. But when they did three base insertions, straddling those inserted residues, they got the frame was restored. So the only possible way that this would happen is if it's a triplet code. They could have done this experiment and still maintain Klingon ease after this. And then when they'd perhaps insert four bases, then if they were able to regain the frame of interpretation, that would tell them it's a four letter code. And so this experiment told them it's a three letter code. So what we know so far, it's a three letter code and it's not overlapping. And so here's some of the data they actually collected. So first, they just looked at the molecular association between trinucleotides and radio labeled tRNAs. And so chemists at that time didn't have all the tools they have today. And they were only able to synthesize trinucleotides of repeating bases. So U, U, A, A, and C, C, C. And so they could put those onto a filter paper and then they put tRNA molecules labeled with a radio label onto there. And they could see basically which tRNAs bind to which trinucleotides. So that allowed them to interpret some of the code. So we know that if it is a triplet code, that there's 64 possibilities. Four to the three is 64. And so we need to figure out, every one of these 64, what is the association between that triplet and a particular amino acid? They continued to this experiment, but perhaps in a more efficacious way, by doing a in vitro translation system. So they took reticulocyte lysates, which have all the enzymes necessary to do translation in vitro. And they fed those lysates artificially synthesized mRNA molecules. And so there's an enzyme, polynucleotide phosphorylase, that's able to synthesize these types of repeating polymers, mRNA molecules, pseudo mRNA molecules. And so for example, they took polyu, and the polypeptide synthesized, when fed to the in vitro translation system, was polyphenylalanine. Polyuc, well they saw serine leucine, serine leucine. So it's u-c-u-c-u-c, u-c-u-c-u-c. And the important thing, when they did these experiments, there's no five prime cap, there's no necessarily a start codon, so there's no signal to start. So all possible frames are interpreted in this artificial in vitro system. Okay, so the ribosome just happens to start wherever it can. But here it's alternating, it's oscillating, u-c-u-c-u-c. So it doesn't really matter where you start, it'll affect like the first amino acid incorporated. But in this example, when you have u-u-c repeating, u-u-c-u-c-u-c-u-c, where you start the frame determines the homopolymer that you synthesize. So u-u-c gives polyphenylalanine, so u-u-c-u-c-u-c-u-c-u-c-u-c-u-c-u, got polyserine, and so forth. Okay, so these are the style of experiments they did, and they could even get more complicated in having four letter repeating units. So this is some of the data from the filter binding assay. What's astounding in this data is the exquisite selectivity that they observed. So u-u-u completely only bound to the phenylalanine tRNA molecule, and there wasn't even a drip of cross or cross reactivity with these other inappropriate tRNA molecules. Okay, selectivity was astounding. So again, here's this data, we have polysingle nucleotides giving polymers of certain amino acids. And as I mentioned here, we're not correctly picking the frame, so where you start determines which homopolymer polypeptide that you actually make, u-u-c. So for example, in this experiment, they determined that u-u-c-u-u-c-u-c-u-c gave phenylalanine, u-c-u-u-c-u-c-u-c-u, gave polyserine and leucine. So they've determined these codons. So they're slowly chipping away at the 64 possible combinations of letters to bring in the amino acid specificity. But then they go to this one, and so the graduate student was doing this, they say, cool, okay, GUA, there's three reading frames, I've done a bunch of these types of experiments. Of course I'm gonna see three different homopolymers of amino acids, he's like, okay, GUA, codes for valine, AGU, codes for polyserine, but UAG encoded for poly-nothing, right? So they got it in the advisors, like, dude, you need to do a better experiment. We all know that we have codons here, but that turns out to be a stop sign. So as soon as the ribosome jumps on in that frame, it just comes off, because that's the signal. There's three different codons that code for stop codons, and that was one of them, okay. So after a while, they put all this together into the book of genetic code, right? So you have a secret code book, and they unraveled the secret, and you really, it's amazing, like why did it have to evolve? This particular association of triplets in each amino acid, it seems somewhat stochastic. What you'll see here is there's necessarily degeneracy, so we only have 20 amino acids. We've got 64 possibilities minus three stop signs, so that's 61 possibilities. So there's gonna be more than one codon, in some cases that code for incorporation of a certain amino acid. Okay, so for example, a leucine, there's a total of four codons, and so they began to, the next question is okay, it's a little beyond our pay grade, why is it like this? One of the reasons we have lots of codons for leucine is leucine is a pretty frequent amino acid and polypeptides. And polypeptides, whatever they've evolved to, need leucines a little bit more than other amino acids, but it's always not necessarily understood, and it could have been stochastic. It's like, hey, this works. We just gotta have some codon that's associated with each amino acid. And so you don't have to memorize this every year. Somebody, this is a critical point, don't memorize this. If you need it on an ASAM, I'll give it to you. I'm cruel, but I'm like, totally evil, I'm just a little bit evil. So these are all the codons, and you can begin to see the degeneracy. Other codons or other amino acids just have a single codon. Like methionine, AUG. And this is doubling up. It's a green, it's a red for stop, green for drive fast. And so that means start and incorporate methionine. We'll see how that works in a moment. Okay, so this summarizes the degeneracy. In other words, the number of unique codons associated with each amino acid here in this table. And so we know that there's 61 total possibilities for 20 amino acids. But how, so we have tRNAs in this very, very specific, this thing base pairs here. So how could we do this? How could we have six codons for some of these? And how could that always be associated with a different tRNA molecule? And what they discovered was fascinating. That there was only, when they started looking at this at the so-called anti-codon down here, they only found generally about 45 or in the ballpark of 45 unique anti-codons. They said, oh, wait a minute, shouldn't there be 61? So if we had Watson-Crick based pairing, yes, there would have to be 61. And so that needed to get figured out. That was a remaining confusion. How can there be less anti-codons? Because each, we know every codon is associated with a certain amino acid. So there must be a tRNA that binds there. So let's take this at a little higher resolution. So here we have a green rectangle with shapes on the end. Let's bring this up. Let's put a little bit more resolution. So now we begin to see the secondary structure of the transfer RNA molecule. And you can see up here in the three-prime end, that's gonna make a covalent attach through an ester linkage, the three-prime hydroxyl, with the carboxylate on our amino acid, okay? And so we have an anti-codon, codon base pairing, anti-parallel. We read the code from five-prime to three-prime in this orientation, and then we're gonna need three-prime to five-prime orientation of the tRNA to make the proper base pairing. So there must be something really magical going on at this interface between the anti-codon and codon to give us the possibility of only having 45 possible anti-codons and still being able to incorporate the right amino acid in all 61 of those possible codons that encode for incorporation of the amino acid. And so here's a little bit more resolution. So we can always think about Watson-Crick base pairing. We know these are RNA to RNA base pairs, right? So we have A to U, not A to T, and we have G to C. And so it's anti-parallel here. But here's a little hint. It turns out that there's other non-traditional, non-Watson-Crick base pairing that can occur at this five-prime position of the anti-codon. So if we have a G or a U there, that can bind to more than one thing in this three-prime position of the anti-codon. Let's look at that in a little more detail. So this was the conundrum. So Crick, he did some important things. He figured out DNA and an Nobel Prize. But he's like, I'm not done, dude, I got more. And then he figured out this, he noticed this piece of data. He's like, wait, 45 unique, 61 needed? And so he came up with this wobble hypothesis. And he did this actually by building stick, ball and stick models of this interface between a tRNA molecule and the codon. And he noticed that that last, that five-prime position in the anti-codon, three-prime position in the codon, there was a little bit of a geometric contortion there. So he began to think, OK, maybe we can accommodate some unnatural type of base pairing in that position because of the geometry of the interface between the codon and the anti-codon. And so his wobble hypothesis, and then they started noticing, now, wait a minute, there's a innocent in this wobble position in the anti-codon and in some tRNAs. And as it turns out, innocent can base pair with A, U, or C. And so I think it's helpful to remember which base is base pair with I. So just think, AUC is AUC. These are the base pairs with I. And so this three-prime position in the codon, five-prime position in the anti-codon was having these nontraditional base pairings. So they say, OK, let's look at these things. So here's our Watson-Crick base pairs, AU and GC. Remember, GC has three hydrogen bonds. AU has two hydrogen bonds. But with inocene, so that's a deaminated adenosine, makes an inocene, right? So remove this amino group. We've got a carbonyl group here. So this inocene could have two reasonably good base pairs, or hydrogen bonds with C, U, and A. There's a bit of a contortion. There's a usual geometry, the positioning of the ribose here. And these are a little bit contorted. But at that last position, because of the geometry of the interface, we could accommodate this kind of base pairing and make reasonably close to linear hydrogen bonds. And the same kind of contortion can occur here. So we have AU base pairs and GC. But you can also have GU. If you have a little bit of flexibility being able to move the U up a little bit. And so in that five prime position of the anti-codon, three prime position of the codon, you can have these types of wobble pairing. So happy wobble, what's wobble? So the first two five prime bases of the codon always form strong base pairs. This was figured out by structural modeling of this. And this provides most of the specificity. But this five prime position can wobble. So if the five prime position in the anti-codon is a C or A, only one codon could be recognized by that TRNA. If the five prime position is U or G, you could have the Watson-Crick and the non-Watson-Crick base pairing. But when the five prime position is an I, you would have three different codons that could be recognized. Remember AUC, AUC. And so here is a more picture, sort of a good summary slide for when you're studying. So we have the Watson-Crick base pairing in the wobble position, a C, G, AU. And then we could begin to wobble U to A, U to G, G to C, G to U, or I to AU or C. And so this helped us to remove or eliminate the requirement that we necessarily had to make 61 unique TRNA molecules. With one TRNA, if it has an innocent there, that could recognize three of the codons. You went for me so far? I'm sorry if I'm going fast. So the other thing that they figured out is when the amino acid is specified by several different codons, the codons that differ in the first two bases require different TRNAs. They did a lot of these experiments. And if the five-prime position in the codon and the middle position change, if there were those changed, you always had a new TRNA because it was generally very tight. There's no wobbling going on in the five-prime position in the codon. But if you have what you can have wobble in the three-prime position. But then if you think about it, in a scene combined three, to bind the next one we need another. So there's, if you just think about two letters, four to the two, 16. And then anything could cover three of the three-prime position of the codon. And then you'd have another nucleotide. So it'd be two times 16 would be the minimal number of TRNA molecules. If every single time you could have an innocent, you did. But that wouldn't work. So if you look at this code, for example, remember, innocent would bind to AUC. We cannot put an innocent here on this TRNA because it spans two different amino acid identities. So say you had a spherogene TRNA with the base pairing with an innocent in the wobble position. Well, then you would get a spherogene with AAA. That wouldn't work. And so I guess evolution is trying to accommodate this necessity to have some, in some cases, a correlation between abundance or frequency amino acids and codon usage. You couldn't always use an innocent whenever you could. Here, you could absolutely use an innocent. UAC could be covered by that with leucine. You would need one more here and then one more here because these differ in the first two positions. With me. This one can wobble, right? AUGU. So this is sort of the process that they use to decode the genetic code. So I think the horse is dead. It beat it to death. Let's move on to a translation machinery. Now you're going to witness perfection. This is so amazing. So let's look at the players. Let's set the stage. So you have mRNA molecules, TRNA molecules ribosomes, and then just amazing assortment of different soluble protein factors that are helping to provide specificity, to provide mechanical forces to push the message along, and to also activate the TRNA molecules. So we have initiation factors, elongation factors, and termination factors. So here we're coming in even a little bit more detail to our TRNA molecule. It has different arms. These arms have names. The D-arm, T-sized C-arm, the anti-codium arm. And what's often referred to as the ACC arm, or the amino acid arm. And so this is an important diagram because it helps you to understand the process of the specificity of the charging, or the addition of amino acid to this TRNA. The pink residues are invariant amongst all TRNAs. So the goal here is to take our TRNA and put only the right amino acid on the right TRNA. And you can imagine, okay, the enzymes that might do this, they could just sort of reach way down at the bottom, tickle the anti-codon and say, okay, I think that's the right one. Or option B, they have a huge potential surface of interaction with this TRNA at all of these green positions. These green positions are variable, okay? So different amino acids that need to be added have different surfaces.  There's also a lot of odd modified nucleotides in TRNA molecules. So for example, a Psi, we saw this last week, that's pseudo-uridine. The D-arm, you might say, why is it D? It looks sort of like a CS show. Well, that's because it has this modified base, dihydro-uridine, and this is the regular uridine. Okay, so the important point here is there's invariant regions, and it makes sense that you would have invariant regions up here, because we want to form an ester linkage with the adenosine, and the factors that are gonna help us to add amino acids to polypeptide, we just have one factor. So it's gotta have something to latch on to. It's gotta be able to predict, oh, this is where the three-prime in is. It's an ACC. So that's important to be invariant. Okay, so that's a TRNA molecule, but really it's a misnamed RNA molecule. It should be called an L RNA, because it's secondary, or it's, I guess you could say secondary structure. It's flat two-dimensional representation is a T. But here in three dimensions, you can see that the D arm and the T-si arm, CIC arm, they were far away from each other, have now folded up on top of each other. And what this is providing is a large armpit. So here's your body, here's your arm, you've got your amino acid, and the enzymes are gonna figure out, okay, I gotta put the right amino acid here. They're gonna actually snuggle up along this whole surface. And all those variable residues will say, mm, I don't think so. I'm not gonna put that amino acid there. So it's important that this be in an L shape, because that provides the maximal number of potential interactions to provide specificity for charging. Okay, so that's the TRNA, the ribosomes. We have differences between bacterial and eukaryotic ribosomes. So they have different large and small subunits, and each of these subunits has different compositions of both non-coating ribosomal RNA as well as proteins. And so, for example, here we have the large subunit bacterial ribosome that has these two ribosomal RNAs and 36 additional proteins. Okay, and here you have one ribosomal RNA and 21 proteins. These are massive, they're 2.7 million Dalton's. You got a lot of proteins, you got these RNA, very, very large RNA molecules, and these are coming together during the process of initiation to translate our mRNA. Okay, and so the other thing to note is we've introduced this nomenclature here, like so in biochemistry class, 30 plus 50 equals 70, right? That doesn't make any sense, because these are actually S, means Fedver sedimentation rate, and the reason that 30 plus 50 does not equal 80 is because this Fedver rate has to do not only with the size of the molecule, but it's cross-sectional area. And if you take two molecules and put them together, you've in effect reduced some of that cross-sectional area from when they were apart. Okay, so if they didn't have this large interface here, they would still have a similar cross-sectional area. And so that nomenclature, so when things get really big, we look at how quickly they sediment in an ultrace centrifuge. And then we have the eukaryotic ribosome, it has its collection of ribosomal RNA, and again, a large number of proteins and slightly bigger, about four million Dalton's. Here's the three-dimensional structure of the ribosome that you might see if you walk by Professor Yogle's office. And so here, the green squiggles and the gray squiggles, those are the actual RNA molecules. And the polypeptides are these sausages. They're like ornaments on a Christmas tree. So back in prehistoric days, maybe 40, 50 years ago, people just could not accept that RNA could be a catalyst. And so they were on this mad quest. We've got like 50 proteins. I'm gonna get a Nobel Prize if I find the one that catalyzes the peptide bond formation. So they went through these one at a time and said, oh, none of them are essential. And when you get this crystal structure, here's three tRNA molecules, one, two, three. And at the interface where polypeptide synthesis is occurring, no squiggles. Ooh, okay, what's going on with this? Here we see the message snaking out. And this is the small subunit down here and large subunit up here. Okay, and so these things are coming together and forming the message. And so there's a larger, much larger number of proteins. So I guess it was reasonable to think, okay, there's more possibility that one of those could be important in the catalysis. There's only one piddly or two piddly ribosomal RNAs, okay? So if you just had this table and nothing else, it'd be hard to see things. But then people started looking at the structure of these ribosomal RNAs and said, whoa, it's so complex. There's so much structure there. Reminds us sort of of the tertiary structure of a polypeptide. And so the naceir said, okay, well that's a very useful scaffold. And that positions the proteins at just the right place. But RNA molecules, yeah, maybe they could do cleav phosphodiester bonds. They sort of know that chemistry or they can transfer phosphodiester bonds between two different molecules. So this was, there were still doubters, haters out there. And so you have this loss of these single proteins that I mentioned this. They took each one of these 30 proteins away and it could always still translate, still make polypeptide, okay? But then they recognized, oh, the trupinosceptor into the tRNA, remember the position that's charged, obviously that's gonna be pretty close to where polypeptide bonds are formed. Well that interacts with, not with a protein, but with a conserved region of 23S ribosomal RNA. And then they started using antibiotics and they say, oh, generally those antibiotics are interacting with ribosomal RNA. Ooh, that's not good. And then they found, okay, I can remove any one of these polypeptides, nothing really happens. If I break a single phosphodiester bond in an inconvenient place within 16S ribosomal RNA, boom, that's it, the thing is completely dead. It's not able to catalyze anymore, okay? And they also noticed, we'll be thinking about something called a Scheindel Garno sequence. So a particular consensus sequence within 16S ribosomal RNA is very important in setting the frame. So it actually base pairs to the messenger RNA molecule. But then they did say, okay, we got rid of the proteins one at a time, we didn't kill catalysis, let's get rid of all of them. And then they said, oh, it can still make polypeptides. So that's, whereas if they removed just one of the ribosomal RNAs, everything didn't work. And then they got the structure, and this is what they saw. Here is the, where all the action is occurring, actually this red position is where polypeptides are made. There's no blue squiggles. And so after a very long decades of argument, people said, okay, it is the RNA molecule that catalyzes this reaction. So let's come back to this thing I mentioned about the setting of the frame. So in prokaryotic, but not eukaryotic translation, the frame is set by binding of the messenger RNA to the 16S ribosomal RNA here and base pairing. So here we have the 16S ribosomal RNA, three prime end of that non-coating RNA is base pairing to very close to the five prime end of the message. So this is the untranslated region of the mRNA molecule. And this base pairing, it was noticed that there's a lot of consensus here and here. So as it turns out, AUG is the start site. And the binding of the 16S ribosomal RNA gets things exactly calibrated. So we initiate translation just at this position at AUG and not UGG or not GAU. It's precisely positioned by base pairing. So here's a variety of different genes. You could say, well, this is not exactly always perfect. Sometimes you have little deviations, but this is the consensus sequence. And so that base pairing is important in setting the frame in prokaryotic. In eukaryotic, it's completely different. Because remember, with prokaryotic transcripts, those are polycystronic. So you necessarily need a mechanism of internal initiation of translation. So a polycystronic message, you have untranslated region functional gene, untranslated region functional gene. So you need to initiate polypeptide synthesis at the five prime end of each of these genes. So the positioning of the Scheindel-Garno sequence allows the ribosome to just drop right into that position. With the initiation of eukaryotic translation, we have a different mechanism where the ribosome lands at the five prime cap because all eukaryotic transcripts have a five prime cap and then it scans until it gets to the first AUG start site. And these are monocystronic. There's no need to have internal just ribosomes landing within the transcript. We can scan, we can't scan up here because we'd have to go through here and the ribosome would fall off at the end because the termination signal also signals the disassembly of the ribosome. So we'd just be making this polypeptide if we had the same mechanism. Does that make sense? Hardest slide to understand today. How did scientists at MIT in the 60s figure out the direction of synthesis of polypeptides and the direction in which messenger RNA molecules are interpreted? Now we know messenger RNA molecules are synthesized from the five prime to three prime direction, but do ribosomes scan five prime to three prime or three prime to five prime? Are proteins made C-terminus to N-terminus or N-terminus to C-terminus? This single experiment answered both those questions. So this system, they used the in vitro, rabbit reticulocyte lysate translation system. They had, they're making hemoglobin. They had a mRNA molecule for hemoglobin and they had so-called polysomes. In other words, more than one ribosome was associated with that transcript at the same time simultaneously and in parallel synthesizing polypeptides. So they let this go for a while in vitro and then after they had pretty good coverage of the ribosomes across the entire transcript, they pulsed in some radio-labeled leucine attached to leucine tRNAs. So at that moment and only at that moment would they begin to incorporate radio-labeled leucines.  Okay, so when they add their radio-labeled leucine, you're near the end here. Leucine is a pretty frequent amino acid in hemoglobin, that's why they picked it. And so they were just labeled the very last part that was synthesized. So they look for the label, where it is in the sequence of the polypeptide, and the label tells them what was synthesized last. So in this polypeptide, they would just have this C-terminus label. But if they incubate this mixture longer, more of the polysomes terminate translation drop off their polypeptide and the label creeps to the left. Okay, so we know from this experiment definitively the C-terminus is synthesized last and that if you give it enough time, this one at the top in 60 minutes can make it all the way through and then you would have label throughout. So the frequency of leucine, it's maybe about every 10 amino acids is a very long polypeptide. So they have pretty good coverage here. So it's really just a stroke of genius, Densis figured this out, it's a Densis experiment at MIT and this told us. And this, once they know the direction that it's in to C-terminus, they could just look at the amino acid sequence, the mRNA sequence have the book of life, the codons, right? And they can then infer the direction of interpretation of the mRNA. So only in the five prime to the three prime direction did they have the right combination that corresponded mRNA molecule codons to polypeptide sequence synthesized. Yes. The red is indicating, and I didn't mention it, I tried to use telekinesis, that didn't work. The red indicates where the label is. Okay, so at four minutes, the label was only found on the amino acids at the C-terminus of the hemoglobin polypeptide. Cause they, remember they're only measuring polypeptides that are released.  So at four minutes, this one's not released, it's still translating. This one was released and they looked at the amino acid sequence and they found that the radio-labeled was at the C-terminus. And so if they give it more time, this is the incubation time with the radio-labeled tRNA, more of these polypeptides that the label would sort of drift to the N-terminus. Okay, cause where the label is depends on where the ribosome was in this process at the moment you added transfer RNA. The radio-labeled, so they set it up so that it was already a polysome. There are just ribosomes all over the place. It's just very simple and absolutely answers the question. Okay, I think I'm gonna move on. So here is a picture of polysomes, isn't it amazing? So here you have DNA and then you have a polysome. I said, ah, what's going on here? There's code transcriptional translation. This is DNA, this line up here. And over here you just have the ribosomes. You're basically synthesizing the mRNA transcript and the ribosomes are just pushing it through the ribosomes, right? They're all piling up here. So only in prokaryotes can you have co-transcriptional translation. Because in eukaryotes, translation and transcription occur in different organelles. So we have transcription in the nucleus, translation in the cytosol. In bacteria there is no nucleus. Ribosomes exist where the transcription is occurring. And so it's probably not an accident that we're interpreting. We're synthesizing the RNA and interpreting it with ribosomes both in the five prime to three prime direction. If we were going in opposite directions we'd have to wait. We wouldn't be able to synthesize polypeptides as quickly, okay?  But here, you could actually have racing. So we'll see in Tuesday's class that ribosomes jump on right away. And if you have a particularly challenging sequence to synthesize through transcription that the ribosomes can start to affect and interfere with the transcription process. And that's actually used in the trip operon. We'll look at that. It's a very, very elegant mechanism that absolutely requires co-transcriptional translation. So beautiful picture of the polysomes. All right, now we're gonna get to the actual steps of translation. You've had this before. Perhaps we'll add a little bit more detail and actually more and more is becoming known about this. So I felt a little uncomfortable with the label they gave to the first stage, activation of amino acids. I think a more appropriate label is charging of tRNA molecules. Converting a carboxylate to an ester, I guess you could, it could be considered activating, but the big picture of what's happening in this first stage is we need to stick amino acids on the three prime hydroxyl of the tRNA molecule. We need to stick the right amino acid. So yes, I suppose we're activated, but really we're charging the tRNA. The uncharged tRNA doesn't have the amino acid the charged one does. We have initiation, elongation, termination and folding in processing. So we're gonna be looking through all these steps. You can come back to this table as we go through this. So step one, the charging of tRNA. And this occurs in two steps by one enzyme. Now each amino acid has its own amino-acylating enzyme. So there's 20 amino acids, there's at least 20 amino acyl synthetases. This one enzyme, remember armpit, snuggle, that one enzyme catalyzes two reactions and it's important that it's a two-step process. First, it activates the amino acid, carboxylate, by ampylating it. So it catalyzes this reaction and in this reaction that enzyme has to specifically find, okay, 20 different amino acyl synthetases, it's gotta find the right amino acid. So it's gotta have a particular binding affinity for that amino acid. Then in the second step, in a different active site, you have the transfer of the, I guess you could say, activated amino acid to the tRNA molecule, the three-prime hydroxyl. So this is a phosphoester linkage, right? And now we have formed an ester. So overall, this is the overall reaction and here's one of the steps, right? So the nucleophilic attack of three-prime hydroxyl on this activated amino acid. So here's the side chain labeled R, amino group, alpha carbon, the carboxylate attached to the alpha carbon is this phospho, I guess you would say, in hydride linkage to our AMP. And so that's gonna make an ester. So we get two chances to check. This is the only time when the translation machinery checks which amino acid is on which tRNA. If we put the wrong amino acid on here, translation will incorporate the wrong amino acid. So we have two-step process where we can check and double-check the identity. We have a nomenclature, the uncharged tRNA without the ester linkage to the amino acid is just called tRNA with a superscript for which amino acid its codon recognizes. And when we charge it, we put serine dash tRNA serous. So this front part indicates that it's charged. There's a ester linkage to the correct amino acid or it specifies the amino acid that's attached. And this part of the name specifies which tRNA molecule we're talking about. Now obviously for leucine, tRNA leucine is not just one tRNA molecule. But the cool thing is, is all the different, I guess there would be minimally three tRNAs for leucine, they all have generally the variable regions of the tRNA molecules are pretty similar yet distinct for that class, for that leucine incorporation. So you could potentially have just one synthetase as recognizing different tRNAs that incorporate the same amino acid. And that's why that variability is important. So we're gonna need 20 different distributions of charges and nucleotides within that tRNA to specifically recognize the amino acids in the synthetase. So this is charging. Here's what it looks like after charging you now have an ester linkage side chain, alpha carbon, amino group on the alpha carbon, carboxylate, ester to the three prime hydroxyl. And so it's very important we need to check and double check here. There's actually two classes of enzymes. Class one's a little unnecessarily complex. The input and output's the same. Amino acid tRNA coming in, out the backside. Amino acid is in an ester linkage to the three prime position. In this class, it initially starts at the two prime position, then moves to the three prime. That detail is not important. And so we have different amino acyl tRNA synthetases for each corporation of each of these amino acids. Here's the structure. The two classes are structurally completely unrelated. One's a dimer and the other's a monomer. This one, the tRNA sort of does the armpit thing. And so all these interactions are saying, oh yeah, that's right, that's right. It puts the right amino acid here at the three prime hydroxyl. Whereas this one, it's actually laying down backwards and it kicks its leg up a bit and it's making a different interaction. That gives us more surface. So we only have variability in certain surfaces, right? And so this gives us optimal specificity. So we've charged the correct tRNA. Now we need to initiate translation. We start with the small subunit. We're doing prokaryotic translation right now. Small 30th subunit has three different binding pockets. E site, P site, and A site. Exit, peptideal, amino acyl site. Right now that means almost nothing, but in a moment it'll mean more. So what we're gonna do first is set things up. Obviously we need to recruit an mRNA molecule. That recruitment is done by that Shine Del Garno base pairing. It's a five prime end of our messenger RNA to the three prime end of the non-coating ribosomal RNA, 16S subunit. So we've recruited our message, but we also need to block some sites. We are gonna initiate by just one amino acid coming in here, okay? And so we're gonna block this E site with IF3 and the A site with IF1. And now we have just one site present and we have a tRNA molecule and it happens to be a methionine tRNA.  So this is just like amazing. It's like what we were doing a peptide synthesizer. Nature has put a formal on the end terminus. Formaldehyde, so H carbonyl is attached to the amino group. And that protects the amino group, okay? And so now we've recruited a tRNA for a methionine and our methionine is up here and this IF2 GTP is checking our work. Only if this base pairing is correct will GTP be hydrolyzed in IF2. Once it's hydrolyzed, there's a conformational change and IF2 is released and then that's cement. There's no going back once you've hydrolyzed GTP. But it takes a while, it's sort of like a clock. Only if the binding is very tight will there be enough time for GTP to be hydrolyzed and for this interaction to be locked in space. The second function IF2 is doing is to make sure the tRNA is charged. So when you think about it, here's a good mission.  We've got lots of tRNAs floating around, one of every type. We've got some that have an amino acid, some that don't. So this thing is checking, okay, we wanna make sure there's an amino acid there because if we just put in the tRNA without an amino acid, you're not gonna be able to synthesize a peptide. Okay, so it has two functions. Proof reading by the clock set by GTP hydrolysis and checking to make sure that this tRNA has is indeed charged. So after that is bound, that GTP hydrolysis drives the disassociation of all of these initiation factors, IFs, as well as recruitment of the large subunit, the 50S subunit. So this is called the 70S initiation complex. So now we have our fMet tRNA fMet in the P site, A and E site are vacant, okay? So next, so here's the initiation factors for bacteria. Eukaryotes are a lot more complicated, but the same ideas are used with a little bit of elaboration. So we still have this idea of blocking the sites that we don't want tRNAs to be binding to, but we also have this snaking, scanning mechanism. So we're not just landing the ribosome by base pairing in the right position. We're landing the ribosome on the five prime cap and then snaking the message through until you get to the right codon, the start codon. So here's a picture of this. So the message is actually wrapped around. The five prime N is attached. It looks sort of like a plasmid. It's not a covalent plasmid, but you have a mRNA molecule, five prime N, three prime N, and these are associated with each other, okay? So you have a five prime cap and eukaryotes, but not prokaryotes. And it turns out you actually do have polyA tails, a little shorter in prokaryotes, much longer in eukaryotes. And so these are linked together.  It's like confusing. So EIF4F is not another polypeptide. It's the complex of these three. It's like, okay, thanks. That's real easy to remember. And so then that we can see here's the ribosome. So EIF3 has a role in recruiting. This is the small subunit of the ribosome. We're at the five prime cap and we need to scan through to this AUG. And this is actually, in the last edition of the textbook, there was no figure. And so now we've learned a lot about how this works. So first we do the same thing as we did in prokaryotic initiation. We block the E and the A site. But then before the message comes in, which is different than what we saw before, we recruit the initiator TRNA. And this is, for whatever reason, it's not formulated. Okay, but it is charged. And EIF2 is doing that surveillance role. So here it's just saying, okay, I'm gonna find the methionine. So IF2 does not bind to like, aspartic acid TRNAs that are charged. It only binds to methionine TRNAs that are charged. And then it brings it into this complex. And then in the next step, we recruit the mRNA that's pre-coated with some of these proteins. And then we're going to literally hold onto the five prime men while we snake the message through. And we're gonna be scanning because we already have the methionine TRNA in the right position. So we have it here. And we're gonna snake the messenger RNA. There it is. There's our start codon. And that will stop only when it has the correct base pairing. And then when that happens, you get GTP hydrolysis, a similar idea. When that binding is stopped and stable enough, GTP is hydrolyzed, and the whole thing just sort of blows apart, leaving us a minimal ribosome where we have an empty E site and an empty A site. So we've got this sort of snaking addition, elaboration of the previous method. Okay, elongation is pretty much the same, prokaryotes and eukaryotes. So we're only gonna look at prokaryotic. This stage occurs through three steps. The binding of the correct amino acid TRNA to the A site, synthesis of the peptide bond catalyzed by ribosomal non-coating RNA and translocation of the message by three, three nucleotides. Okay, so here is the first step. So we have the empty A and the E in the A site. And now we figure out, oh, that's why you call it the A site, because that's where the amino-acillated TRNA lands. The first amino-acillated TRNA is the only TRNA that lands in the P site. Okay, all the others, every amino acid we're gonna add, we're gonna put in the A site. So here's our second base pair.  So we have EF, elongation factor, TU, GTP, binds. Doesn't really check the identity of the amino acid. It maybe does, but we don't really have good data on that. But it does make sure that it's charged, okay? And what's happening here is all the possible 20 TRNAs, complex with EF, TU, GTP, are sampling in the site. So you have potentially like 19 other TRNAs come in there and they're just sort of coming in and out. When they have the correct base pairing, they stop. And they stop long enough for GTP hydrolysis. And so they're checking this interaction, being very careful, using energy. So this is really just using for specificity. We're spending energy to make sure it's the correct polypeptide. So here we have the binding. And when this binds, and when GTP is hydrolyzed, the TRNA is like this, and it goes like this. It's like walk like an Egyptian. So here it is pointing out here, and then it switches. And the reason it can do that is this big honking EF-TU gets out of the way. So now it's like, oh, whoa, hey, here's another amino acid. Or an ester-linked amino acid over here. And now these two active sites are directly positioned. So this is important. So if it's the wrong amino acid, TRNA, coming in here, this is nowhere near this. So even if it samples, as long as it doesn't pivot, we're not gonna get the wrong amino acid. So this is sort of a blocking function as well. So now we've got the two amino acids nearby to each other, both ester-linked. Okay, and so now we're gonna have the amino group and the A-site of the second amino acid in this case. Nucleophilic attack on this ester, kick the electrons up, back out the figure in your textbook. Oh, the shame of the way the electrons are moving. I mean, it's just, don't even look at it. It breaks your heart to see. But here you have, it doesn't just kick up. That's a transition state. It kicks out the ester and moves over here. So here you have the effluent amino acid here and attach a new amide bond formed. And this is the amino acid in the second position. Here's the side chain. There's a methionine in the first place. And now we're at the three-primohydroxyl in this A-site. But now, to continue, we need to vacate the premises, so we need the amino acyl site to get the heck out of the way so another tRNA can come in. And so we have a conveniently shaped, so here's tRNA with the EFTU and here's this EFG GTP. That thing looks the same. And basically it's like, lands without codon specificity, it lands and bumps this thing out of the way.  And that motion requires GTP hydrolysis. This is not really a proofreading, a checking step. This is a literal movement of the whole mRNA and the associated tRNA. So now you have the first tRNA in the E-site. We now know why it's called the E-site, it's the exit site. And then we have the peptide, the nascent peptide in the peptidyl site. And we have EFG here. Once its GTP is hydrolyzed, it moves things out of the way and then it takes off and now we have a vacant A-site. And so we repeat this process for every amino acid incorporated. All right, so elongation we saw now that it proceeds through three steps. What about termination? Well, termination, you get to that stop codon and remember sampling all possible tRNAs. So UAGs, we're waiting, expectantly, for the UAG anti-codon tRNA to come in and bind there. It never happens. Instead, there's a protein that comes in, a release factor. Again, shaped similarly to a tRNA, except for there's a little extra space up here. There's a little pocket of water that's in here. And so now, when we bind here, it's in the right orientation to stimulate the peptide bond formation activity. But we have no amino group here. We don't have a charged amino acid. So instead, we make a new bond to water. There's a little bit of a water pocket. And so now that hydrolyzes off the polypeptide. It drifts away. And then in an active process, we disassemble this. So we use EFG, GTP. Remember that was moving the message along and that can also have the role of sort of pushing this apart and disassembling this process. There's also this ribosomal release factor. We have a name, but we don't really have much mechanistic detail on that one yet. So that leaves us ready to go. We already have one of our initiation factors that's blocking the e-site. So we're ready to go another translation, okay? So overall, we have for each, for the elongation stage, for each amino acid incorporated, we're using five, the hydrolysis of five high-energy phosphate bonds. So remember the charging, each tRNA that comes in. We had to use the cleavage of ATP to AMP. That's two phosphodiester bonds. And then here that we had proofreading and then EFG, GTP is moving the transcript along, okay? And then we know we had one GTP for initiation. Remember it was bound to our tRNA F met, the IF2, I want to say, okay? So here's all the steps that we've seen. We've terminated and now we can go to the last part. And this is sort of, this gets into the, remember the lipid lecture, how painful that was? Just here's this and this and this. There's all kinds of different modifications. You can modify proteins in the N or C termers. As it turns out that methionine almost always cut off as the thing is being translated. So that methionine, there's a peptidase waiting to cut because why should we have to have a methionine on the N termers? So if you look in genomic databases, you always see a methionine at the first position. It's not there in the polypeptide that's made. It's lopped off, okay? And so we can have whatever amino acids we want. We have to start with methionine because that's the start code on. But then we lop it off, no restrictions on amino acids. We have signal sequences, that's important localization, variety of modifications, disulfide bonds. We've seen these throughout the semester. This is a summary, we know too much about phosphorylation of alcohol containing amino acids. Remember these are changing the charge. You have a nonpolar to a charged residue can change shape, change activity, change binding interactions. Carboxylation, here's glutamate being carboxylated or lysines can be methylated. I mean this goes on, there's literally hundreds and hundreds of different ways you can slice and dice modifications of these amino acids post-translationally. We've looked a lot at glycosylation, adding glycosyl or polysaccharides to various amino acids. We know this occurs in the ER. It's important in targeting and providing unique epitopes on proteins that are exposed to the surface, the outside environment. We also might, some of you might know that various proteins are lipidated. So, RAS, many of you will be clinicians. So, RAS is important in cancer. And so, RAS protein can either be in the cytosol or it can be post-translationally lipidated with this cholesterol building block, right? Remember this, it can be lipidated at a cysteine and that lipid is a nice anchor which will take the cytosolic RAS and keep it associated with the cellular membrane. Okay, so this is just a lot of different stuff. Let's see. Right, okay, so there's lots of antibiotics. So, antibiotics are useful for killing organisms and for studying translation.  All of you would probably be dead or a lot of you would be dead without these antibiotics that specifically target prokaryotes, so bacterias. When you were young and you got an infection, you might have had molecules like erythromycin, streptomycin, tetracycline. These are critical and they have exquisite selectivity for the prokaryotic versions of the ribosome. Other ones of them are less fortunate. They can be deadly toxins to humans such as, you might have heard of ricin or diphtheria toxin and so these specifically inhibit eukaryotic translation. But some of them sort of hit both.  I don't think it's a bad idea. I'm gonna talk about one in detail. So if I were to ask you questions, I might ask you, okay, does it hit eukaryotic or prokaryotic, are there certain antibiotics that hit eukaryotic prokaryotic? Yes, true, circle T. Pyromycin, I'm gonna show you how that works. So then you might wanna pay a little bit more attention. So here's pyromycin. What does it look like? What does it look like? So a little hint, we've got a peptide in the P site. This pyromycin antibiotic lands in the A site. What does it look like? What is this? Sort of like adenosine-ish, right? Adenosine-ish, it's got this thing, not adenosine, but it's structurally similar to adenosine. You have arrivos, remember this is the three-prime in, aminoacyl site, and then you have, well, not an ester linkage, you've got an amide bond, just something amino acid-ish, right, little greasy. So this thing, obviously, normally what's binding here is a massive tRNA molecule, and at the end is this little aminoacylation. And so we're mimicking the end, but there's nothing to interact with the codon. There's no anti-codon-codon interaction. So this fits in this active site, and we're not gonna make, we are gonna make an amide bond. Yeah, so we're gonna actually make an amide bond to this, but when we make an amide bond to this, it's not bound by anti-codon interaction, it just goes away, and it prematurely releases the polypeptide and actually just sort of stays there and kills the ribosome. It takes a catalyst that could go on and synthesize new polypeptides and completely kills it. All two million Dalton's of that molecule now needs to be disassembled, okay, because it's completely, it's a suicide inhibitor, okay? So that's pure mycine, you might wanna know it a little bit about that guy. Okay, so we have protein targeting is important. We have internal signal sequences are important, or targeting the proteins for export through the ER, or in bacteria don't have an ER for export out of the plasma membrane. We also have interminable signal sequences for targeting to mitochondria chloroplasts. Other sequences and signals within polypeptides for localization are internal. So generally the ones that are on the interminous, once you get to the right location, don't need them anymore. They put artificial constraints on the sequences in the polypeptide, so those are generally cleaved off. The internal targeting sequences are not cleaved off. So proteins targeted in the nucleus have that nuclear localization signal stay within the sequence. So we're gonna look at the ER, targeting of ER to the ER. So here you have a highly greasy functionality with a few positive charges. So all proteins that are targeted to the ER, either for export from the cell or incorporation into cellular membranes have this particular signal sequence. Then what happens is the interaction of, so you begin to synthesize this polypeptide. There's a reason the signal sequence is on the interminous, because as soon as you start to make that signal sequence, a SRP protein binds that signal sequence, interacts with the ribosome and says stop. That's enough. What we're about to make, because generally things that go into the ER are greasy. If we just made those completely in the cytosol, you get all kinds of damaging aggregation of proteins. We want to stop that. As soon as we see that signal sequence, stop and then bind to the ER. So we have an SRP receptor that binds. We snake the nascent polypeptide that's still being translated through a channel, translocation complex. And this formation of this complex leads to a release of the SRP. And as soon as SRP is released, the ribosome says, okay, I'll keep going. Releases, it extrudes like a noodle through this channel, through the ribosome into the ER. And then a peptidase, not related to the release of SRP, there are peptidases in the ER that just cleave off these signal sequences, because we don't need that. Okay, so this is perhaps for export from the cell. We just extruded in there for placing proteins in the membrane. I actually added this slide pretty late because I was just curious, how does that happen? So there's both a signal sequence that targets the ER, recognized by SRP, but then there's two additional sequences, a signal anchor sequence and a stop transfer anchor sequence. And so in this, we're not really sort of landing on the ER and extruding our polypeptide into the ER. We bind, associate here, but then we sort of step off a little bit from the ER and make a little bit of sequence. And these sequences are recognized by this particular protein. So first, you have these two. And when you make the second alpha helix, obviously these are gonna be right charged or uncharged hydrophobic amino acids tangentially from the axis. And so when we finish synthesizing this alpha helix, that's a signal to this translocase dissolve. Go away. And when that happens, we end up with those two alpha helices in the membrane. We make another two. It's recognized by this binding complex. When it recognizes that second alpha helix, dissolve, go away. And so we're doing these two alpha helices at a time. And we end up with our integral membrane protein. I just hope that was pretty cool. Okay, so if we make proteins, we need to be able to degrade them. I know this lecture's a slog. Record number of slides. And so many of you know about ubiquination. So ubiquin is a 76 amino acid polypeptide. It's attached through its C-terminus to the epsilon amino group of target proteins. So there's a series of enzymes, or classes of enzymes, E1, E2 and 3. E1 activates the ubiquitin using ATP hydrolysis. So you attach the ubiquitin to a cysteine and the E1. And then the action, and then E2, you transfer the ubiquitin to the E2, again, cysteine. Now E2, the combination of E2 and E3 provides both substrate ubiquitin ligase activity and substrate specificity. So we wanna ubiquinate the correct proteins. E3 is more specificity, E2 is more of the ligase. And there's all kinds of classes of these. At the end of the day, your target protein has 76 bizarro amino acids hanging off the epsilon amino group of its amino acids. And so these signals are recognized by the proteasome, binds and degrades the protein. So this is a way that we can actively signal the turnover of proteins. As it turns out, remember we cut that methionine off at the end-terminus? So we revealed something surprising. And what we revealed is correlated to how long that protein will last in a cell. If we reveal any of these amino acids, that protein will generally last 20 hours. Whereas if we reveal an arginine at the end-terminus, that protein's gonna be degraded in about two minutes. So there's some, and we're still trying to understand, okay, something's gotta be recognizing what's going on at the end-terminus, looking for certain amino acids, and then interacting with the ubiquination system to be able to degrade the protein. So protein turnover is an important regulated process. So after the clicker, for those who are geeky like me, we have the Paul Berg movie on translation, hippie translation. There were no drugs involved in this process. Yes. So we also have a question for the audience. Okay, one quick question, guys. Maybe it's the clicker question. Perhaps. It goes back to the dentsus experiment. Yep, the dentsus experiment. Maybe I asked a question on the dentsus experiment. Yeah? Go ahead. If amino acids at the C-terminus are indicated by the red markings, how does the dentsus experiment indicate that the protein is made from end-to-C-terminus? Because in the dentsus experiment, the labeling indicates not what was made first, what was made last. So in the dentsus experiment, they saw the label first at the C-terminus, and so that was made last. Because the polypeptides that were released very quickly were almost already done with translation before you added the radio label. Okay, so that's a confusing experiment. I'm hiding, I'm hiding here. You're gonna lynch me. How are you guys doing? Okay, I'd like to get to our entertainment part of today's lecture. Has everybody voted? A lot of words necessary to convey a very simple meaning here. Everybody voted? Any more votes coming in? A few more? Click, click for your lives. Okay? So it's C. All right, we're gonna, movie time, who brought popcorn?  Molecular happiness. You're going to have that opportunity for this film attempts to portray symbolically, yet in a dynamic and joyful way, one of nature's fundamental processes, the linking together of amino acids to form a protein. We know now that the three dimensional structure and the function of a protein is determined by the order of amino acids to form the back of the molecule. So protein synthesis involves programming and assemblies. And this film, with people portraying molecules using the dance idiom, tries to animate these two processes, the programming and assembly of a protein. Our genes carry the instructions from ordering the amino acids of each protein. Those instructions are encoded in the message of RNA, depicted in this film for the long, snaking chain. Each of the message units is played by three adjacent people in the chain. Color head balloons indicate the basis. Green foquani, blue for uracil, yellow for adenine. If there is a message, there must be a way to translate that message. And that's the job of the ribosome and of the transfer RNAs. The ribosome is composed of a large and of a small subunit. And these are depicted in the film as tumbling, rolling clusters of bodies, amorphous by themselves, but organized and structured when in the act of translating the message. First, the small subunit with the aid of an initiation factor captures the message. Then the first transfer RNA carrying its cognitive amino acid is brought to the ribosome message complex by a second dancing initiation factor. This requires energy and that's represented by a puff of smoke. Next, a large ribosomal subunit tumbles into place and then the process of bringing each amino acid through the ribosome message complex is accomplished by the t-factor and its GTP cohort. And so in the order prescribed by the balloon colors and the message chain, each amino acid is brought to the assembly site to be added to the growing chain by the peptidio synopase. Next, in an energy requiring step, the message RNA, tRNA complex is shifted so as to bring the next message unit into the ribosome to allow the process to repeat itself. At the end, the terminator factor, seeing the termination signal, cleaves the completed protein from the last tRNA releasing it from the ribosome. So that the ribosome can do its job again, the two subunits are split apart and separated from the messenger by the ribosome releasing factor. My diagram is a necessity static but protein synthesis is a dynamic process. This movie tries to bring those dynamic interactions to life. It was brilliant and the slimy 30s ribosome did gyre and gimbal in the way message unit around. Initiator factor two went searching for tRNA who bore the placid amino acid.


I'm currently studying Deterrence and Compellence and the International Security Course here at the War College. I think it prepares me with the tools necessary and understanding of what deterrence is and how I can use it within my warfare area as an information warfare officer. Deterrence allows you to see our information environment is changing where we need stability and consistency. We also need to be able to identify resources that put us in a position to be able to deliver a psychological effect that deters others from taking hostile action against us. I think this is building that environment and the resources and capability that this course has provided me gives me a thorough understanding that I can move forward with and be ready to take on the next challenge.


So I spent a lot of time talking to you all on my Discord, Facebook group and on consulting calls and I've noticed a pattern. A lot of you are very proud of your artistic depth but are concerned that an audience won't get what you're all about with just one song. And with good reason, one of the things we discuss so often is that in a playlist dominated world the audience really doesn't form a relationship with you or music unless they go deeper. So many of you want to release bigger releases instead of just a single to show your audience your depth and try to form a deeper relationship with them. Even worse, many of you can't figure out how to get them to simply follow you on socials or Spotify or to stay in touch with you. So in this video, we'll discuss how to build relationships with fans after they hear your music. Hi, I'm Jesse Cannon, a music marketing nerd who's teaching musicians how to grow their fan base from zero to ten thousand fans and this is Muse Formation. So I know sometimes musicians think that the only way an audience will like them is if they see all their depth and talents and that can stem from their own self-consciousness or often delusions as they think their songs that have influences of Blink-22, Newfound Glory and Yellowcard just won't be appreciated unless the world cares how diverse their influences are. But aside for the fact that musicians are, surprise, surprise, way too far up their own ass, it can often be true that if you've worked hard on making your artistic persona filled with a ton of depth, it can help you connect with fans and get people talking about you if you show them more of that depth. A few weeks ago, I had a great talk with Charlie Harding of the amazing podcast Switched On Pop which you should really be listening to. Anyway, he wrote this article for Vox that's a must read and is linked in the description about how the big complaints of musicians today when they blow up on playlists is they can't make the jump to having fans who actually care about them as opposed to just listening to that one song they like on the playlist. The fans don't buy merch or come to the shows which I know a lot of you are concerned about now that we're back to trying to play live again. But truthfully, this isn't new and I've been having conversations about this for at least three to four years. So I've had a lot of time to observe which artists make the jump from being just another song on a playlist to an artist that has fans who are actually engaged with them. Let's talk about why many of the artists don't become an artist all these fans who clearly like their music are engaged with. The most common reason that a fan doesn't stay engaged with you is you don't feed them. Here's an example. I keep this playlist I call Now Now Now on Spotify and it's basically every song I've loved for the past five years from an artist who doesn't have an EP or an album that I can listen to. So I throw them on this playlist and then listen when I want to hear a shuffle of songs I actually enjoy. If I looked through this playlist and tried to figure out why my relationship died with some of these artists and sure enough the number one cause was often close to a year until they would put out another release and since I've talked to tons of friends about this subject over the years they say the same thing. I imagine you even probably feel the same way which is yet another reason to utilize consistent sustained promotion. If you don't know what I'm talking about I highly suggest you watch the playlist on the screen or click on the link in the description below.  But there's obviously more to it than just hoping for the best with curiosity. I mean obviously you want to make things easy for your fans and link your socials and your Spotify bio. You did that right?  Anyway one of the reasons we talk so much about telling stories on this channel is they give your fans something to tell their friends about you but stories also inspire curiosity that keep fans thinking about you and talking to their friends which is why this is so important since the more you're on their mind the more likelihood the relationship is going to keep growing. We have to remember these stories give you the chance to show that artistic depth that you want people to see in you. They can finally see why your friend group should have a reality show or see how much you think about your music or understand what a thoughtful and interesting person you actually are. Whatever it is the stories you tell on social media and in your visuals are how you get past the gap of a fan who listens to you on a playlist and doesn't think more about you and one who does. Since if you aren't telling stories and they check you out they have no reason to follow you especially in a world where they aren't staying tuned to get merch drops or see when you're touring. We have to remember at some point if you're always telling stories and getting other fans excited about you they'll see your name on social media and dig deeper. So it's so crucial that you tell these stories in a consistent and sustained way or you're never going to convert fans from casual listeners. If you don't know what I'm talking about watch this playlist on how to tell stories around your music which is linked on the screen now or in the description. Let's also remember one of the principles we often discuss is the only way fans talk about you is if you do eventful things and if you just make the same video as everyone else where you wrap up the camera in a warehouse in some shitty supreme gear well that's not eventful it's just what all the other fuckboys do. Eventful things are the stories you tell but they also are the conversation pieces that get fans talking about you. The way I often think about music videos or shows or any other marketing we're going to do in a marketing plan is I think about what the fan would tell their friends about it are we doing something so eventful that they'll want to tell their friends and then I would think about what they would say about your marketing move and then I work backwards from there. This technique is somewhat stolen from Amazon was a mandate that you can't ask for a meeting at the company for a product unless you write the press release for what Amazon would announce about it in advance so everyone could think of how to make the product as eventful as possible. But we're talking about relationships here right? Well yes I've told you a thousand times that the consistent sustained promotion is the main way to make that happen but so many of you are impatient and want people to understand you from the jump of your project and while that impatience I think lacks a lot of logic we will discuss that in another video but because I'm a really thoughtful and nice youtuber I'm going to indulge you for a minute here. So we discussed that you need to marry an eventful promotion that people will find remarkable enough to tell their friends with a strong story where people will get to know you well enough they will form a relationship with you where they will think about you more and become a casual fan. So if you're trying to do this I have one example of something that I keep seeing employed more and more. One of the most eventful things you can do is a big narrative video where you tell a longer story or string a bunch of songs together in video form. I like to call these videos a big splash in that since they are remarkable you can make this big splash people turn around to see as opposed to when you release regularly you have more times people may see the ripple you're making since you're spreading it over time throwing rocks in the pond and if you're doing it often enough that last stone is still rippling you could throw another one in the pond and it'll make the last ones rippled even more. The long form video though makes this big splash which sometimes can make the huge difference of people taking you seriously and helps solve the problem people are going to see this as really eventful as opposed to just another music video. This occurred to me recently as two different artists who were on my radar put out these longer form videos and suddenly I became a huge fan of both of them and now I'm totally obsessed with them and they took me from someone who'd maybe give a listen here and there to them to now listening non-stop and texting my friends to check out both of these artists. The first was the hardcore group Turnstyle who I've probably given a cursor you listen to on each of their releases but they never really hit me since when I hear hardcore cliches I'm brought back to sitting through tons of terrible sets of boring hardcore acts all through my teens. I mean I didn't think I would ever even like a hardcore record again but damn what Turnstyle's done here is wild and while it's undeniable that Turnstyle are writing some of the catchiest songs in the genre while also being innovative and has way more depth than anything I've heard since Refuse made The Shape of Punk to come I wouldn't have realized this if I just heard one song but since they released this four song video called Turnstyle Love Connection with transitions in between each song I was able to hear their artistic depth and become more curious and intrigued. I was able to take in how much artistic depth they had and coupled with the impressive visuals I kept coming back to this video again and again and it got me absolutely hooked on this band which is even more impressive as the singer directed the whole thing and if you watch the video it really is impressive. The group telling a story coupled with an incredible visual made me excited to share it with everyone I know who would enjoy it. Now I should say this this was the perfect time for a group like Turnstyle to do this. They've long been that band with the great buzz and doing decent numbers but this is not one of those steps that will cement them in the upper echelons of the genre but since we discussed promoting when you have zero to ten thousand fans on this channel it's important to make the distinction that this wasn't a technique to make them do that type of jump but instead solidify them as a genre leader. So this is a good time to talk about coming out of the gate with these larger statements. Tierra Wack came right out of the gate with her video and album Wack World almost as a mission statement and what made it eventful was a really well-directed 15 minute video with 15 songs all under a minute which as TikTok was beginning to boom got tons of press trying to diagnose this as a trend that we'd all be writing shorter songs for TikTok and if memory serves me right we may have even been calling it bite dance then or something else. Anyway I think that trend was forecasted a bit wrong as songs have needed to exist both in TikTok and outside of it and the fact is one thing hasn't changed about songs in 70 years is that hearing a chorus three times seems to be the most effective way to deliver songs into people's brains so it sticks there. I remember reading an actual scientific study on this in the book Stuck on Repeat and we see over and over again that repeating a hook three times is a law just like gravity. It can occasionally be defied under the right circumstances but most of the time it's going to be the law. Okay but back to Tierra while she seemed to have a solid music business team she also had this hypothesis that this new form of expression in the songs would get people talking and with whatever team she had behind her it really seemed to work as I really heard so many rumblings about her and it made a splash that got her on some pretty sizable features for an artist right out of the gate. The next artist I want to discuss is the other one I recently built a relationship with and was calling all my friends about. Shy Girl who put out the four song big splash video blue. In it she shows the artistic depth a different sound she can do as an artist. Now Shy Girl is one of those artists with a decent buzz with under a dozen songs out already and only one EP out and her numbers really weren't that huge and until this video I feel like she was bubbling under with a little bit of a buzz and was an artist people didn't take as seriously but has now cemented herself as a force to reckon with and one that people are really talking about and there's tons of examples of people doing these types of videos. A bit before her unbelievably sad passing Sophie put out a few new songs with a long form visualizer set called Heaven Suspended which kind of seemed like a repurpose DJ set she did since she didn't get to play it out enough live. I saw this material being DJ'd right before the pandemic but it was an eventful mid-pandemic drop that generated tons of buzz and further submitted her as one of the most interesting artists out there. Quadeca who put out some songs I love this year put out his record from me to you with a full album video and I feel like Quadeca was an artist people weren't taking very seriously and then he did a major turn of perception by showing a ton of artistic depth including being smart enough to caption the full video to show off his lyrical prowess and show the viewers how much different artistic depth he had compared to just being another YouTuber rapper. And let's be real here this is why eventually putting out an album after a while is still necessary for a lot of artists to build relationships with fans since if they like your album it's a much longer relationship building exercise since having someone's listened to 12 songs in a row is literally 12 times more of a relationship builder than a single song but more about that some other time. So unless you have an insane budget you do miss out on CSP keeping you in the conversation when you do these big splashes but there's ways around this. I've even seen shy girl release a whole other video treatment for the breakout song from blue BDE which features the Buzzy UK rapper Slowtie and the video allows her to cash in on some further CSP after generating some buzz from the initial splash of blue but really I know a lot of you want to jump out of the gate with a big splash like this but one thing to keep in mind is if you have no platform it could be hard for this to really work well for you since people need to hear about it. One thing all these rs we just discussed have in common is they had some sort of leg to stand on they were ascending up to a bigger level so many of you will ask about doing this for your first songs and it honestly will 99 times out of 100 be a lot of work for less reward than if you already had some buzz and then put out a big splash. So what my rules for doing a big splash would be is this. Make sure you have an infrastructure for people to see it. Do you have publicity and the right people lined up in a team where if you make an amazing big splash project it will get seen? Is this the time in your career where you can really take you to a new level by doing a huge statement? I would also have a CSP plan for after you make that big splash so you don't drop the ball. You need to keep feeding an audience in these days and if you do that and use CSP after a big splash it's going to go all the more further. Since I don't think these videos are the only route to people taking you seriously and understanding you and most artist's careers are built off consistent sustained promotion today where they let people see their depth and growth over time through stories on social media and their music videos. Let's also remember the majority of artists you've loved in your life have not done these big splash videos but the concept here of doing something like that where you really make a big splash and do a profound statement at some point really can help lift you some levels and that's why I find this worth discussing for smaller ours. Big splashes allow fans to take in a lot of you and build a relationship that keeps them interested in you but there's tons of other ways to do it and the key is to remember to tell stories that show your artistic depth and personality in an eventful way and that will make your promotions more effective and hopefully get the attention of an audience that will see you and jump from that playlist. All right if you enjoyed this video please send it to a friend and discuss it as well as liking and subscribing and getting notified since on this channel we're always discussing things like this. If you want more there's a video on the screen now that talks about how to get your music noticed as well there's also a video on how to go from zero to 10,000 fans and another one on how to promote your music on Spotify. Thanks for watching.


California convening a special climate summit will bring together all of the different groups that play out. California is not waiting for Trump. We know that you can talk the talk, but now it's time to... Our message to local leaders needs to be inescapable. No more stalling, no more delays, it's time to act. This September 8th, let's show world leaders what real climate leadership looks like. Real climate leadership means guaranteeing good jobs to active workers and communities. Real climate leadership means no new fossil fuel anywhere. Building a new economy for all. Leaving no one behind. 100% renewable. We're organizing the biggest climate mobilization that the West Coast has ever seen. The whole world is invited to rise up in their own communities. Rise up for what's local. Rise up for what you love. I'm rising on September 8th because I believe that our voices should be heard and heeded by the leaders meeting for the Global Climate Action Summit. You're rising because of South Africa to show true climate leadership. I'm rising because the Pacific will show the world what true climate leadership is. Rise for climate, jobs and justice. I rise. I rise. I rise up because I rise.