BIOCHEMISTRY DISCUSSION 10

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All right! So here we are at the last chapter in the lecture series, and it’s a big one - protein synthesis. This is a big deal, so let’s get on with it.

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Biochemistry: A Short Course

Fourth Edition

CHAPTER 40 The Mechanism of Protein Synthesis

Tymoczko • Berg • Gatto • Stryer

“Protein synthesis is the major task performed by living cells.”

That is a very strong statement! But it’s made by David Goodsell, who is one of the people that I work with at The Scripps Research Institute, and I think that he has probably the best understanding of what cells do out of anybody I've ever met. He says - “for instance roughly one third of the molecules in a typical bacterial cell are dedicated to this central task,” which is protein synthesis. So that’s more molecules than are dedicated to DNA replication, or transcription, or glycolysis, or any other function that the cell performs. Protein synthesis is the biggest job cells have. So I'm going to say you should look at this video first - I've provided you a link. And this video is a very good representation of protein synthesis. It’ll give you all the general ideas - but do be aware it is a very simplified representation. Things don’t go smoothly in the world of the cell. They don’t flow chunk, chunk, chunk, chunk like that. They are banging around with each other. There’s a ton of random interactions that occur, and you're not going to see that in this video. But this video will give you a great overall picture of what’s happening in protein synthesis - and so please look at it now and then come back and finish this slide. Ok – now that you have watched the video, just remember that the tRNAs don’t fly directly toward the active site and line up like they do in that video. They are running around - and inappropriate tRNAs are delivered to active site 20x as often as the right one is. So it’s important to know that there’s a lot going that is not being pictured - and you won’t see that in ANY representation, really, of this process. It’s kind of sad, and it’s one of the things I plan

on doing in my career, is developing some more videos that represent the stochasticity of this environment. So you can imagine the A site being open and molecules are bombarding it constantly, water molecules and other proteins, and glucose, and all kinds of stuff - and other tRNAs as well. And the tRNAs actually have to hit in the correct orientation also, so that they will get bound to that site. Now, you can imagine that among all of the molecules in the cytosol it would take a lot of collisions for the correct tRNA to find that codon at the A site - and I think that it would probably take, I mean, literally like a trillion different collisions before the right thing got in there. But we’re talking about this on a molecular scale – and on a molecular scale, collisions are taking place about a trillion times per second, so a trillion things, that’s a huge number, I mean, that’s like almost incomprehensible, a trillion things are hitting that active site, the A site, every second, let’s just say, this is approximate, I don’t know the actual numbers, but probably not that far off. And it turns out that the rate of protein synthesis is actually about 6-9 amino acids per second - so yes, it takes a long time (or a lot of collisions) for the correct one to hit, but they’re hitting so fast that it actually is producing proteins at a rate that we can understand.

All right - so let’s break down what we’re going to talk about in specific terms in this chapter. First we’re going to talk about protein synthesis decoding the information in the messenger RNA, and that’s your general recognition of the tRNA. Then you’ve got your peptidyl transferase catalyzing peptide bond synthesis - that is a lovely reaction. Peptidyl transferase actually is an RNA enzyme, which is kind of interesting. There’s a difference between bacteria and eukaryotes in the initiation, and also differences in other steps of protein synthesis, and those differences are very important. We’re going to talk about bacteria first, like usual, because it’s slightly simpler, and then we’ll talk about why these difference are so important. Then we’ll talk about inhibition of protein synthesis and why that can be useful. And also secretory proteins - there’s a slightly different mechanism that occurs when you’re trying to make a secreted protein. And finally we’ll end up with regulation of protein synthesis.

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Chapter 40: Outline

40.1 Protein Synthesis Decodes the Information in Messenger RNA

40.2 Peptidyl Transferase Catalyzes Peptide-Bond Synthesis 40.3 Bacteria and Eukaryotes Differ in the Initiation of Protein

Synthesis 40.4 A Variety of Biomolecules Can Inhibit Protein Synthesis 40.5 Ribosomes Bound to the Endoplasmic

Reticulum Manufacture Secretory and Membrane Proteins

40.6 Protein Synthesis Is Regulated by a Number of Mechanisms

Alright. So we’ll break down protein synthesis into three main parts: initiation, elongation, and termination. If I recall correctly I think that’s pretty much how we broke down DNA replication and transcription as well. So the first step is going to require ribosomes, tRNA, mRNA, and some special proteins called initiation factors, and the second two steps require a slightly different suite of molecules.

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Section 40.1 Protein Synthesis Decodes the Information in Messenger RNA

Learning objective 3: List the key components of the protein-synthesis machinery.

Learning objective 4: Describe the roles of RNA and proteins in protein synthesis.

• Protein synthesis consists of three parts: initiation, elongation, and termination.

• Initiation requires the cooperation of ribosomes, tRNA, mRNA, and proteins called initiation factors.

Figure 40.2 An active ribosome. This schematic representation shows the relations among the key components of the translation machinery.

First of all we’re going to go into some overall anatomy of the ribosome and the tRNA and mRNA complex. There are 3 tRNA binding sites on the ribosome, and they are partially connected to the 30S subunit. The 30S subunit is where the mRNA and tRNA interact, and that’s the decoding part. And then the 50S subunit catalyzes the attachment of the amino acids to form a protein. So there are 3 sites for binding of tRNA to the complex, and they are labeled 1) the A site, the aminoacyl site where the incoming aminoacyl-charged tRNA arrives, and 2) the P site, or the peptidyl site – that’s where the growing peptide chain is attached. There's a tRNA there and it’s actually attached to the whole peptide chain. And then the final site, is 3) the exit site, where, after the peptide chain has been transferred to the next tRNA, the deacylated tRNA (without the amino acid attached to it) remains attached the ribosome in that position, in the exit site - and that’s primarily just to hold mRNA in place. We’ll talk a little bit about that later. The tRNAs at the A and P sites come together up at their 3 prime terminus, where the amino acids are attached, and that’s where the peptide bond is formed. And adjacent to that site is a channel that allows the growing peptide chain to exit the ribosome through the back.

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Ribosomes Have Three tRNA-Binding Sites That Bridge the 30S and 50S Subunits

• The acceptor stems of the tRNAs in the A site and P site are near one another at a site on the 50S subunit where the peptide bond is formed. A channel connects this site to the back of the ribosome through which the peptide exits the ribosome during synthesis.

• There are three tRNA binding sites on the ribosome. At each site, the tRNA is in contact with both the 30S subunit, which holds the mRNA being translated, and the 50S subunit.

1. The A (aminoacyl) site binds the incoming tRNA.

2. The P (peptidyl) site binds the tRNA with the growing peptide chain.

3. The E (exit) site binds the uncharged tRNA before it leaves the ribosome.

Figure 40.1 Binding sites for transfer RNA. (A) Three tRNA molecules are shown in the tRNA-binding sites on the 70S ribosome. They are called the A (for aminoacyl), P (for peptidyl), and E (for exit) sites. Each tRNA molecule contacts both the 30S and the 50S subunit. (B) The tRNA molecules in sites A and P are base-paired with mRNA. [(B) Drawn from 1JGP.pdb.]

Here’s the diagrams that we have in the text of this. And you can see from these diagrams how the three tRNAs are side by side, in the E site, P site, and A site, and bridging between the 30S and 50S subunits of ribosome. On the right hand side we can see where the tRNA is attached to the mRNA. That’s taking place in the 30S subunit, the smaller subunit. And you can see that there is NOT an interaction depicted at the E site between the mRNA and the tRNA, but we’ll talk about that in a bit. I think there should be.

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Diagram of the Binding Sites for Transfer RNA

Here are just some profiles of David Goodsell drawings of the players in this first step. We have the messenger RNA down there at the bottom, and the transfer RNA up at the top with the little blue amino acid stuck to it. Blue, like the other blue parts of the ribosome small subunit, which are also protein. And the ribosome small subunit is a mixture of protein and RNA, ribosomal RNA

And this diagram is an example of what a growing amino acid chain might look like. It’s just a simple string of amino acids in a line, but once they exit the ribosome they’ll fold up into that globular shape that you see there on the right. Notice also they kink at the bottom of this string of amino acids, because of the presence of the proline.

Figure 40.2 An active ribosome. This schematic representation shows the relations among the key components of the translation machinery.

So here is the first of the diagrams that the book is going to use to illustrate this process - and I have some problem with this, especially with regard to the E site. I believe that the E site tRNA is still attached to the mRNA, but we’ll talk about that a little more in a bit. This does show pretty well the incoming tRNA with its amino acid stuck to it, in the A site, and then the P site containing a tRNA with 2 amino acids, and those are starting to go out the polypeptide channel at the top of the 50S subunit. The A site is the aminoacyl site, because it brings in single amino acids. The P site, the P stands for polypeptide, because that’s where the polypeptide chain is attached. And then the E site is the exit site, which is a final binding place of the tRNAs before they leave the complex.

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Diagram of an Active Ribosome

Here's another view of that, and in the center portion the surrounding amino acids and RNA have been removed so you can clearly see the tRNAs and amino acids in the center. Once again, the tRNA that is attached to the growing protein strand is at the P site, and the new tRNA with its amino acid in green is attached at the A site

So we’re going to talk about bacterial translation first, and in fact I think that all of the diagrams so far are taken from bacteria. But there are some similarities of course between bacterial and eukaryotic. The start signal for both, for example, is AUG. And that is the first codon in the translation of a protein. That codes for methionine. (And I guess GUG can also be used, but that’s probably more of a bacterial thing.) But of course you also need some other bases to help identify that start site - you know, you can’t just start at any methionine - and you need to have some bases that code for the termination site as well. So pretty much any mRNA that you have that codes for a protein will have special sequences that tell the translation where to start and where to stop. Remember we talked about polycistronic mRNA when we discussed the lac operon - when the lac operon is being expressed, it produces a single RNA that codes for 3 separate proteins. But each one of those proteins does have its own start sequence and termination sequence. So they are not translated all together. Sometimes proteins are translated all together and then cut into smaller pieces afterward, but we’re not going to talk about that right now.

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The Start Signal Is AUG Preceded by Several Bases That Pair with 16S

Ribosomal RNA (1/2) • Many mRNAs in bacteria are polycistronic, meaning that a

single mRNA encodes for multiple proteins. Each of the coding regions has its own initiation site. The first codon to be translated is usually AUG, which codes for methionine.

• Sequences in the mRNA denote where initiation occurs and where synthesis stops.

So initiation in bacteria starts close to the 5’ end of the mRNA, but not right up to it - it’s got to be about 25 nucleotides away. And the sequence that the ribosome is looking for to find the start site is called the “Shine-Dalgarno” sequence, named after the people who discovered it. And the 30S subunit of the ribosome will find that sequence. It interacts directly with the 16S ribosomal RNA, which is a part of the 30S subunit and you can see that at the bottom in green - there’s a slightly highlighted green section. That is the 16S rRNA that is binding to the red mRNA Shine-Dalgarno sequence.

When the 30S subunit is bound to the Shine-Dalgarno sequence, it positions the AUG codon in the P site to align for the translation of the very first amino acid.

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The Start Signal Is AUG Preceded by Several Bases That Pair with 16S Ribosomal RNA (2/2)

• Initiation in bacteria begins at least 25 nucleotides from the 5’ end of the mRNA. The nucleotides between the 5’ end and the first codon translated, an example of an untranslated region (UTR), contain information, called the Shine–Dalgarno sequence, that directs the protein synthesis machinery to the start site.

• The Shine–Dalgarno sequence is a purine-rich sequence approximately 10 base pairs upstream of the start site that interacts with the 16S rRNA to correctly position the AUG codon in the P site.

http://pdb101.rcsb.org/motm/121

Figure 40.3 Initiation sites. Sequences of mRNA initiation sites for protein synthesis in some bacterial and viral mRNA molecules. Comparison of these sequences reveals some recurring features.

Now, here are a spectrum of Shine-Dalgarno sequences that you can see. They do differ basically based on organism - so small changes in the organism’s ribosomal RNA will cause it to recognize slightly different Shine-Dalgarno sequences. And even within a single organism, such as E. coli, the Shine-Dalgarno sequence might be different for different proteins, and the differences in the Shine-Dalgarno sequence may have an effect on how frequently that protein is translated, or the rate at which its translated, or the timing of the translation - there are a number of variables that can be altered by changes in this sequence.

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Diagram of the Initiation Sites

Now, the very first tRNA that gets included in this process is a very special one. It’s called formylmethionyl transfer RNA, and it’s got a methionine on it, but that methionine has got a formyl group stuck to the N terminus, which would be the very beginning of the protein chain. This formylmethionine is special, and is recognized by different proteins than your regular methionine tRNA. The shape is different enough so that the formylmethionine tRNA will not bind to other AUG codons in the mRNA.

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Bacterial Protein Synthesis Is Initiated by Formylmethionyl Transfer RNA

• A modified form of methionine, N- formylmethionine (fMet), is the initiator amino acid in most proteins in bacteria.

• It is activated by attachment to the initiator tRNA called tRNAf. f-Met-tRNAf binds only to the initiation codon (AUG) and not to AUG elsewhere in the mRNA.

• Another tRNA—tRNAm—recognizes internal codons for methionine.

• The same synthetase activates both tRNAm and tRNAf, and a specific transformylase modifies the methionine attached to tRNAf.

Figure 40.5 Translation initiation in prokaryotes. Initiation factors aid the assembly, first, of the 30S initiation complex and, then, of the 70S initiation complex.

A series of initiation factors assemble at the junction there with the mRNA and the 30S ribosomal subunit. Initiation factors 1 and 3 - we are not going to talk about what they do specifically, but they do prevent the 50S subunit from binding prematurely to the 30S subunit, which you don’t want to have happen. You need to get the formylmethionine in there first. And the formylmethionine comes attached to initiation factor 2, which is a GTPase. So, probably in conjunction with other proteins, this formylmethionine tRNA and IF2 (Initiation Factor 2), that complex is the one that finds the spot on the 30S initiation complex and delivers that first formylmethionine tRNA to the AUG start site. The 50S subunit binds, and the GTPase, the initiation factor 2, hydrolyzes its GTP, thus making this reaction very unlikely to proceed in reverse. And the other initiation factors leave, and the 50S and 30S subunits come together around the formylmethionine tRNA complex in the P site - and that is your initiation complex.

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Formylmethionyl-tRNAf Is Placed in the P Site of the Ribosome in the Formation of the 70S Initiation Complex • Initiation factors (IF) assist in the assembly of the

protein-synthesizing machinery.

• IF1 and IF3 bind the 30S subunit to prevent premature binding to the 50S subunit.

• IF2, in cooperation with GTP, delivers fMet-tRNAf to the mRNA, which is already correctly positioned on the 30S subunit by the Shine–Dalgaro sequence, to form the 30S initiation complex. Structural changes lead to the ejection of IF1 and IF3. IF2 stimulates association with the 50S subunit

• The 50S subunit binds, leading to the hydrolysis of GTP by IF2 and departure of the initiation factor, forming the 70S initiation complex. The fMet-tRNAf occupies the P site bound to AUG, thereby establishing the reading frame.

So you’ve got the 70S initiation complex surrounding the formylmethionyl tRNA, which is in the P site, but the A site is empty. So at this point new aminoacyl tRNAs are able to enter in that A site. Now, it takes a little bit of energy to get that aminoacyl tRNA into that site, and that energy is supplied by elongation factor Tu, which is a special protein, that’s also a GTPase, that binds to the aminoacyl tRNAs out there – whatever’s floating around, they bind to this elongation factor Tu, and then together they diffuse and start falling into the A site. If the anticodon that is with the tRNA pairs with the codon that is in the mRNA, then the elongation factor Tu hydrolyzes its GTP, using that energy, and dropping the correctly matched aminoacyl tRNA into the A site. Now, remember that elongation factor Tu does not interact with the formylmethionyl tRNA, because that is interacting with initiation factor 2 (that’s a number 2 instead of the little Tu. I don’t know why they do that, but it does help to distinguish between them). So initiation factor 2 and elongation factor Tu actually play a very similar role, which is that of delivering the tRNAs to the proper locations and then hydrolyzing GTP to get them inserted.

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Elongation Factors Deliver Aminoacyl-tRNA to the Ribosome

• In the 70S initiation complex fMet-tRNAf occupies the P site and the codon for the second amino acid is exposed in the vacant A site.

• Elongation factor Tu (EF-Tu), in association with GTP, delivers the appropriate aminoacyl-tRNA, as specified by the codon, to the A site. If the anticodon pairs with the correct codon, GTP is hydrolyzed, and EF-Tu-GDP departs with the A site now occupied by the appropriate aminoacyl tRNA.

• Elongation factor Ts (EF-Ts) induces release of GDP from EF-Tu, which is replaced by GTP, and another cycle can begin.

• EF-Tu does not interact with fMet-tRNAf.

A P E

So now we’ve got our two aminoacyl tRNAs sitting there next to each other, and it’s time to bring those amino acids into proximity and form a peptide bond. That is catalyzed by the 23S rRNA, in an area called the “peptidyl transferase center” on the 50S subunit. If you’ll recall, the amino acid is attached to the 3’ end of the tRNA by the ester bond to its carboxyl end. So the amino terminus is floating around, and when it gets into that active site, they put it in the right position, right next to the other amino acid, so that the amino terminus can attack the carboxyl terminus of the amino acid in the P site, at which point it releases the tRNA that it’s attached to, and now you’ve got a peptide bond formed between the 1st and 2nd amino acids.

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Section 40.2 Peptidyl Transferase Catalyzes Peptide-Bond Synthesis

http://www.cell.com/cms/attachment/589273/4514274/gr5.jpg

Learning objective 5: Explain the role of the ribosome in protein synthesis.

• Peptide bond formation, an exergonic process, is catalyzed by a site on the 23S rRNA, called the peptidyl transferase center. The 23S rRNA is a component of the 50S subunit.

• Some of the catalytic power is due to catalysis by proximity and orientation.

• The aminoacyl-tRNA in the A site makes a nucleophilic attack on the ester linkage between the tRNAf and the formylmethionine molecule in the P site.

• An eight-membered transition state forms. Collapse of the transition state yields peptide bond formation and an uncharged tRNA in the P site.

A P E

So at this point, you’ve got an empty tRNA in the P site, and you’ve got the tRNA with the growing peptide chain that’s actually coming up that middle section, but the tRNA is still in the A site. So another elongation factor, elongation factor G, we also call it translocase, comes along and basically pushes that whole assembly one codon in. This moves the tRNA that was in the P site over to the exit site, and the tRNA that was in the A site is now in the P site. It takes the hydrolysis of a GTP to power this reaction. But now we have gotten everything back to the starting position. And we can continue to elongate - just repeat the process. So the polypeptide chain grows from the amino terminus to the carboxyl terminus, with the incoming amino acid amino termini attacking the carboxyl termini of the previous growing polypeptide chain.

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The Formation of a Peptide Bond Is Followed by the GTP-Drive Translocation

of tRNAs and mRNA • Upon peptide bond formation, the growing chain

is in the P site of the 50S subunit while attached to the tRNA in the A site. The tRNA in the P site of the 30S subunit is uncharged.

• Elongation factor G (EF-G), also called the translocase, uses the energy of GTP hydrolysis to translocate the mRNA by one codon.

• Upon translocation, the peptidyl-tRNA is fully in the P site, the A site is vacant, and uncharged tRNA is in the E site, disengaged from the mRNA.

• The polypeptide chain grows from the amino terminal to the carboxyl terminal.

A P E

Figure 40.9 Mechanism of protein synthesis. The cycle begins with peptidyl-tRNA in the P site. An aminoacyl-tRNA binds in the A site. With both sites occupied, a new peptide bond is formed. The tRNAs and the mRNA are translocated through the action of elongation factor G, which moves the deacylated tRNA to the E site. When there, the tRNA is free to dissociate to complete the cycle.

So let's just take a look at this diagram, which illustrates the beginning of the peptide chain formation. And in the very first panel, there on the upper left, we have what would be the formylmethionine there at the P site. And then the next aminoacyl tRNA comes in to the A site, bound with elongation factor Tu. And then the peptide bond is formed by the attacking of the amino acid in the P site by the amino acid in the A site. And we can see on the lower left this actual reaction in a lot more detail than you see in the book. In blue, in the center, you see the 3’ end of the tRNA in the P site; up at the top you can see the ribose of the terminal adenine, and you can see, attached to the 3’ oxygen, still in blue, you have the peptide chain. Now, the incoming amino acid is in red, and the nitrogen on the amino terminus there will attack the end of the peptide chain. You can see in green a lot of residues from the surrounding proteins that are involved in this, and also, in grey, you see water molecules that are involved. Another part that's involved is the 2’ hydroxyl group on the ribose, and interestingly I think this might be why DNA can't do this job - because it doesn't have that 2’ hydroxyl group. So, after the new peptide bond is formed, the elongation

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Diagram of the Mechanism of Protein Synthesis

http://www.cell.com/cms/attachment/589273/4514274/gr5.jpg

factor G will bind and hydrolyze a GTP, and that energy will be used to push the entire assembly one spot down. And now your aminoacyl tRNA, with the peptide chain attached, is in the P site, and the A site is empty and ready to receive the next aminoacyl tRNA. Now, this shows the empty tRNA dissociating before the new tRNA comes and binds the A site, and I don't think that's true. I think that the tRNA will bind the A site before the empty tRNA dissociates.

Here’s a slightly more correct (I think) representation of this whole process. And we’re looking at the elongation cycle – and, just in particular, I’d like to just point out that the E site remains populated until after the A site is filled. Now, I actually dug around quite a bit to see if I could get some real hard evidence about what happens, and in what order, and I was kind of surprised to find that actually it’s still an active field of investigation. People are not quite sure exactly when the tRNA leaves the E site. But one thing that did seem to be clear is that there are always two tRNAs attached to the mRNA, at least. And that’s important, because those two tRNAs hold that mRNA in place, and prevent it from slipping. Imagine if you had a string of uracil in your mRNA, it would be really easy for that to slip down one, you know, one base. And that would screw everything up, because then you would be out of frame, and the rest of the protein would probably be non-functional. So you need to have two of those tRNAs there to hold it in place to prevent slippage. When the new tRNA comes in to the A site, it actually induces a conformational change in the ribosome, which then allows the tRNA at the E site to diffuse away.

Figure 40.11 Polypeptide-chain growth. Proteins are synthesized by the successive addition of amino acids to the carboxyl terminus.

Well, a vastly simplified diagram, basically just showing that you’ve got your tRNAs attached to your amino acids. And tRNA2 comes in, and then the 1st amino acid gets attached to that, and then tRNA3 comes and your 2nd amino acid gets attached to that, and that’s how the peptide chain grows.

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Diagram of Polypeptide-Chain Growth

Figure 40.10 Translocation mechanism. In the GTP form, EF-G binds to the EF-Tu- binding site on the 50S subunit. This binding stimulates GTP hydrolysis, inducing a conformational change in EF-G that forces the tRNAs and the mRNA to move through the ribosome by a distance corresponding to one codon.

And here is a very poorly drawn diagram (I think) of elongation factor G. It comes in and hydrolyzes the GTP, and that causes a conformational change, which pushes the mRNA down one codon.

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Diagram of the Translocation Mechanism

This is a much more realistic picture of what the elongation factor G might look like (on the right), and also the elongation factor Tu (there on the left). So you can see, on the left, the elongation factor Tu, bound to a tRNA, will float into that particular spot. And if it fits, if it fits perfectly, then it will trigger the hydrolysis of a GTP that is bound to elongation factor Tu, and that will release the tRNA, with its attached amino acid, and also induce a conformational change in the 30S subunit, which will rotate that tRNA so that it is right next to its growing polypeptide chain in the P site. The new peptide bond is formed, and then elongation factor G would drift into that spot and hydrolyze a GTP, and the energy there will be used to shift the entire assembly down one spot so that the tRNA is now in the P site entirely.

Figure 40.12 Termination of protein synthesis. A release factor recognizes a Stop codon in the A site and stimulates the release of the completed protein from the tRNA in the P site.

Now, for the termination phase, there is a stop codon. There are 3 stop codons in humans - UAA, UGA, and UAG. And those do not pair with any tRNA. Those pair with a special protein called a release factor (RF). So when the stop codon arrives in the A site, it hangs out until a release factor comes in. When the release factor comes in, it goes into the active site and causes the polypeptide chain to be released from the tRNA that it’s attached to, and it’s free to go - and now it’s a protein. At this point, with the release factor stuck in there and no more polypeptide chain, the ribosome has finished its job and doesn’t need to do anything else, and it dissociates with the help of EF-G and ribosome release factor, so that it can reform at another start site and start translating another protein.

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Protein Synthesis Is Terminated by Release Factors That Read Stop Codons (1/2)

• The process of elongation continues with the growing chain exiting the ribosome through the channel in the 50S subunit until a Stop codon—UAA, UGA, UAG—appears in the A site.

• Stop codons are not recognized by any tRNA but are recognized by proteins called release factors (RFs).

And here is a better illustration of what RF1 would actually look like sitting in the A site.

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Protein Synthesis Is Terminated by Release Factors That Read Stop Codons (2/2)

http://pdb101.rcsb.org/motm/121

RF1 4v4r

• RFs recognize Stop codons and facilitate the attack of a water molecule on the ester linkage between the polypeptide chain and tRNA in the P site, releasing the polypeptide chain.

• EF-G and ribosome release factor (RRF) catalyze the dissociation for the ribosome, mRNA, and attached tRNA in a reaction facilitated by GTP hydrolysis.

A P E

http://pdb101.rcsb.org/motm/121

So that’s prokaryotic protein synthesis. And eukaryotic synthesis is very similar, but the ribosomes are larger, and protein synthesis starts with a normal methionine instead of a formylmethionine. There is a special tRNA that is associated with that normal methionine, and that tRNA is required for the initial formation of the initiation complex, but that’s an important difference that we’ll discuss in a bit. In eukaryotes there is no Shine-Dalgarno sequence. The ribosome starts to form at the 5’ end of the mRNA, which you may recall is marked with a cap that makes it so that it can be specifically recognized. The…40S ribosomal subunit finds that 5’ cap, and then scans to find the very first AUG in the sequence. As usual, there are a number of protein initiation factors that are involved, but we’re not going to go into depth on that. Another interesting difference is that in eukaryotes the mRNA complex is circular. Proteins that bind to the 5’ cap and the poly A tail interact with each other, thus bringing them together and making it a circular assembly.

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Section 40.3 Bacteria and Eukaryotes Differ in the Initiation of Protein Synthesis

Learning objective 6: Compare and contrast bacterial and eukaryotic protein synthesis. • Although the basic plan of protein synthesis is the

same for all organisms, eukaryotic protein synthesis is more complex in a number of ways.

1. The ribosomes are larger, consisting of 40S and 60S subunits that form the 80S ribosome.

2. Protein synthesis begins with a methionine rather than formylmethionine. A special initiator tRNA called Met-tRNAi is required.

3. The initiator codon is always the first AUG from the 5’ end of the mRNA. More protein initiation factors are required.

4. The mRNA is circular because of interactions between proteins that bind the 5’ cap and those that bind the poly A tail.

Elongation and termination are very similar except for that bacteria have two release factors, and eukaryotes only have one. I don’t know why that is, but that’s the way it is. Protein synthesis occurs in the cytoplasm in eukaryotes, and RNA synthesis occurs in the nucleus. So remember, that’s the other very important difference between prokaryotes and eukaryotes, is that eukaryotes have a nucleus, so things need to be transported between the two (cytoplasm and nucleus). And protein synthesis can not take place until the mRNA has been exported from the nucleus into the cytoplasm.

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Eukaryotic Protein Synthesis is More Complex

5. Elongation and termination are similar in eukaryotes and bacteria except that bacteria have two release factors but eukaryotes have only one.

6. Protein synthesis occurs in the cytoplasm, whereas RNA synthesis occurs in the nucleus in eukaryotes. Moreover, protein synthesis machinery is organized into large complexes associated with the cytoskeleton.

Figure 40.13 Eukaryotic translation initiation. In eukaryotes, translation initiation starts with the assembly of a complex on the 5ʹ cap that includes the 40S subunit and Met-tRNAi. Driven by ATP hydrolysis, this complex scans the mRNA until the first AUG is reached. The 60S subunit is then added to form the 80S initiation complex.

So here’s a diagram of eukaryotic initiation, and you can see that it all starts at the 5’ cap. The 40S subunit of the ribosome binds there, and it looks like it has the methionyl tRNA associated with it already. And, of course, there are a ton of initiation factors and other things that are not pictured here. The 40S subunit, with its attached methionine, begins to move down the mRNA, and that movement is powered by ATP hydrolysis. And it continues moving until it reaches an AUG codon, which is probably recognized by the methionyl transfer RNA. When that happens, the initiation factors detach, and the 60S subunit attaches, and you’ve got yourself an active translation complex.

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Diagram of Eukaryotic Translation Initiation

Figure 40.14 Protein interactions circularize eukaryotic mRNA. [After H. Lodish et al., Molecular Cell Biology, 6th ed. (W. H. Freeman and Company, 2008), Fig. 4.28.]

A couple of interesting things here. We see at the 5’ terminus of the mRNA (with the cap on it), you have an eIF-4E protein attached there, and an eIF-4G protein attached to that. These are two of the things that are part of the initiation complex. At the other end, the 3’ end, you’ve got the poly A tail, and you have two poly A binding proteins that are attached to that poly A tail. The poly A binding proteins associate with the eIF-4G, and make the whole complex circular. You also see 3 complete ribosomes moving down the mRNA, translating the protein. And yes, that can happen…as soon as the initiation site (the AUG) becomes free, another ribosome can form on it, and start to translate, so you’ll have multiple proteins being translated simultaneously.

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Diagram Depicting how Protein Interactions Circularize Eukaryotic mRNA

Inner Life of the Cell

So - antibiotics, a lot of them, they inhibit protein synthesis. And because of the differences between bacterial protein synthesis and eukaryotic protein synthesis, antibiotics can target bacterial protein synthesis without affecting eukaryotic protein synthesis - which is exactly what you want! Streptomycin interferes with the formylmethionyl tRNA complex, so that it can’t bind, and therefore it will prevent protein synthesis in bacteria. But we don’t use formylmethionyl tRNA in our bodies (in humans/eukaryotes), so it doesn’t affect us at all. So there you go - that’s a way to target the bacteria only, without bothering the other tissues. Of course, there are ways to inhibit protein synthesis in both, and puromycin is an example of a chemical that does that. Puromycin causes the release of uncompleted polypeptide chains - you know, maybe it’s some sort of like, termination factor analog - but that will prevent proteins from being properly synthesized.

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Section 40.4 A Variety of Biomolecules Can Inhibit Protein Synthesis

CLINICAL INSIGHT Some Antibiotics Inhibit Protein Synthesis

• The ability of many antibiotics to inhibit bacterial protein synthesis while leaving eukaryotic protein synthesis unaffected makes them powerful therapeutic agents.

• Streptomycin interferes with the binding of fMet-tRNAf and thereby inhibits protein synthesis initiation in bacteria.

• Puromycin inhibits protein synthesis in both eukaryotes and bacteria by releasing uncompleted polypeptide chains from the ribosome.

Other antibiotics that you might have heard of, that are involved with the inhibition of protein synthesis, are tetracycline, erythromycin, chloramphenicol, and cycloheximide. So there are a ton of these guys, actually, that that’s what they target. And if you can screw up protein synthesis, then you will screw up that organism - because, as we said in the beginning, protein synthesis is the main job that occurs in organisms.

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Table 40.1 Antibiotic Inhibitors of Protein Synthesis

Here are some diagrams of tetracycline, on the left, bound to the small subunit of the ribosome - and that blocks the site where the tRNA binds, so you can’t get tRNA in there, you can’t translate any protein. Chloramphenicol, on the right, is blocking the site on the large subunit that the amino acid gets added to - so the site that catalyzes that peptide bond formation. And of course, if you can’t make peptide bonds, then you can’t grow proteins.

Now, once you’ve made a protein, that protein needs to get to the right place in the cell to do its job. The majority of the cytoplasmic proteins will just float around, banging into everything, until they find the right place to stick. But what about proteins that would be transmembrane proteins, or secreted proteins, or proteins that you want to get into a specific organelle such as a lysosome or the Golgi apparatus? Well, there's a separate mechanism for translating these types of proteins and it's essentially the same - the mRNA floats around, the ribosome assembles, and it begins to translate - but after it’s translated a little bit of the protein, there's a specific sequence which targets that protein to the endoplasmic reticulum, and the ribosome will stop translating until it gets to the endoplasmic reticulum, at which point it will translate the rest of the protein INTO the endoplasmic reticulum. And when you have these ribosomes attached to the endoplasmic reticulum that's what we call the rough ER (the rough endoplasmic reticulum) because it looks like it's covered with stuff.

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Section 40.5 Ribosomes Bound to the Endoplasmic Reticulum Manufacture Secretory and Membrane Proteins • In eukaryotes, protein sorting or protein targeting is the process of directing

proteins to distinct organelles such as the nucleus, mitochondria, and endoplasmic reticulum.

• Two pathways are used to sort proteins. In one, completed proteins are delivered to the target.

• In the other pathway, called the secretory pathway, proteins are inserted into the endoplasmic reticulum membrane cotranslationally.

• Protein synthesis in the secretory pathway occurs on ribosomes bound to the endoplasmic reticulum (ER). ER with ribosomes bound is called the rough ER, or RER.

Figure 40.19 Ribosomes bound to the endoplasmic reticulum. In this electron micrograph, ribosomes appear as small black dots binding to the cytoplasmic side of the endoplasmic reticulum to give a rough appearance. In contrast, the smooth endoplasmic reticulum is devoid of ribosomes. [Don W. Fawcett/Science Source.]

And here you can see in this electron micrograph, the rough endoplasmic reticulum looks like, you know, a series of lamellae. Remember these are two-dimensional kind of sacs that are going around (like layers of an onion) – we’re just looking at a cross- section. And they are covered with these little black things - well, those little black things are ribosomes. Ribosomes are so large that they are one of the only complexes that you can see clearly in electron micrographs.

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Electron micrograph of Ribosomes Bound to the Endoplasmic Reticulum

So, for these secretory proteins, they begin their synthesis in the cytoplasm, just like any other kind of protein does. But at the N terminus (at the very beginning of the protein) is a special sequence that forms a structure that identifies it as a secretory protein. When that signal emerges from the ribosome, it stops production until the ribosome comes in contact with the endoplasmic reticulum. And then when it does, the protein synthesis starts again, and now the protein is being pumped directly into the endoplasmic reticulum.

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Protein Synthesis Begins on Ribosomes That Are Free in the Cytoplasm

• The synthesis of proteins bound for the secretory pathway begins on ribosomes that are free in the cytoplasm.

• Once a portion of the nascent protein that contains a specific signal emerges from the ribosome, synthesis is halted and the ribosome complex is directed to the ER.

• Once bound to the ER, protein synthesis is reactivated, with the nascent protein now directed through the membrane of the ER.

So those initial sequences that come out of the ribosome are called the signal sequence. And when the signal sequence comes out, those are hydrophobic amino acids, so they form a little blob - and that blob is recognized by a protein called the signal recognition particle (the SRP). It’s a GTPase, and it binds to the signal sequence and stops the progress of the ribosome until it gets to the ER.

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Signal Sequences Mark Proteins for Translocation Across the Endoplasmic

Reticulum Membrane (1/2) • Several components are required for the cotranslational

insertion of proteins into the ER.

1. The signal sequence is a sequence of 9 to 12 hydrophobic amino acids (sometimes containing positively charged amino acids) arranged in an α-helix, often located at the N-terminal region of the primary structure, that identifies the nascent peptide as one that must cross the ER membrane. A signal peptidase in the lumen of the ER may remove the signal sequence.

2. The signal-recognition particle (SRP), a GTP-binding ribonucleoprotein with GTPase activity, binds the signal sequence as it exits the ribosome. SRP binding halts protein synthesis and directs the complex to the ER.

At the ER, this protein (the SRP) docks on a receptor (the SRP receptor), which is attached to the membrane of the ER. Now the ribosome, with its emerging protein, is pretty much stuck to the outside of the ER, and floats around until it reaches a hole, which is a protein conducting channel called a translocon. And when it finds that hole, then it feeds the end of the protein through that hole and starts the protein synthesis again, with the protein passing through that hole and into the interior of the ER. When the SRP-ribosome complex reaches that hole, GTP is hydrolyzed and the signal recognition particle and its receptor are able to dissociate, and go start to do some work with some other ribosomes.

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Signal Sequences Mark Proteins for Translocation Across the Endoplasmic

Reticulum Membrane (2/2)

3. The SRP receptor, an integral membrane protein with GTPase activity, binds to the SRP-ribosome complex.

4. The translocon, a protein-conducting channel, accepts the ribosome from the SRP-SRP receptor complex, and protein synthesis begins again with the protein now passing through the membrane in the translocon.

• Upon GTP hydrolysis, the SRP and SRP receptor dissociate and begin another cycle.

Figure 40.20 The SRP targeting cycle. (1) Protein synthesis begins on free ribosomes. (2) After the signal sequence has exited the ribosome, it is bound by the signal- recognition particle (SRP), and protein synthesis halts. (3) The SRP-ribosome complex docks with the SRP receptor in the ER membrane. (4) The ribosome–nascent polypeptide is transferred to the translocon. The SRP and the SRP receptor simultaneously hydrolyze bound GTPs. Protein synthesis resumes, and the SRP is free to bind another signal sequence. (5) The signal peptidase may remove the signal sequence as it enters the lumen of the ER. (6) Protein synthesis continues as the protein is synthesized directly into the ER. (7) On completion of protein synthesis, the ribosome is released and the protein tunnel in the translocon closes. [After H. Lodish et al., Molecular Cell Biology, 6th ed. (W. H. Freeman and Company, 2008), Fig. 13.6.]

So here’s the picture. On the left you’ve got your mRNA, and your signal sequence emerging from the ribosome. And the signal recognition particle attaches to the signal sequence and stops the mRNA’s progress through the ribosome. It’s not very clear how it happens in this picture, but we will get to it in a second. The SRP binds to the SRP-receptor in the membrane, and that brings it to the translocon. And when the GTP is hydrolyzed, the translocon gets opened and the protein gets fed through into the ER lumen. At this point, a signal peptidase can cleave that signal sequence off, since its only function was to guide the protein to the ER. It’s not necessary for the function of the protein afterward, so it can just get rid of it. The protein is

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Diagram of the SRP Targeting Cycle

synthesized directly into the ER, where it folds, and after it’s done the ribosome can detach and get ready to do its job again. This diagram shows a single mRNA passing through several of these ribosomes, which I guess is actually possible, so you know, no big deal. But probably not terribly likely.

Now, this is an animation of the whole process that I think is very good, but I wasn’t able to insert it into the presentation. So please pause the presentation now and follow the link to the narrated version on YouTube of this animation.

So let’s talk a little bit about the regulation of these processes. Once you’ve created a messenger RNA, you can now make proteins off of it, but that doesn’t necessarily mean that you WANT to make proteins off of it. It’s good to have around, but sometimes you might want to wait and make proteins at a later time. Sometimes you might want to stop, and stop making proteins altogether, and degrade the mRNA. That’s the standard way to stop making proteins off of an mRNA, is just to destroy the mRNA, and then it can’t make any more proteins. So, for an example, we are going to talk about how iron regulates the translation of several proteins. Now, iron is very important, because it’s important for obviously carrying oxygen through your blood, and also as a part of the electron transport chain, but it can also be destructive, so you have to be very careful with it. The regulation of iron in the cell is mediated by a number of proteins, including transferrin, which transports the iron, the transferrin receptor, which binds the transferrin and lets it get into the cell, and ferritin, which is a way of storing the iron inside the cell. Now, you would like these proteins to be regulated by the amount of iron that is in the cell, much like the lac operon is regulated by the amount of lactose that is in the cell. Well, in the lac operon, we control that by controlling transcription, but with the case of ferritin and these other elements, we’re going to control the production of these proteins at the translational step. So ferritin mRNA will form structures, just like any mRNA would. One of these structures is a stem-loop, which occurs in the 5’ untranslated region. That is the region between the 5’ end and the start site for translation. And this stem-loop structure is called the iron response element (IRE). And it binds to a protein called the

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Section 40.6 Protein Synthesis Is Regulated by a Number of Mechanisms

Learning objective 7: Explain how protein synthesis can be regulated.

Messenger RNA use is subject to regulation.

• Iron is a key component of many important proteins, including hemoglobin and cytochromes.

• However, iron is also capable of generating destructive reaction oxygen species, so iron transport and storage must be carefully regulated.

• Important proteins in iron metabolism are transferrin, a blood protein that transports iron; transferrin receptor, a membrane protein that binds iron-rich transferrin and facilitates its entry into the cell; and ferritin, an iron storage protein in the cell.

http://pdb101.rcsb.org/motm/35

Transferrin (top, with iron bound) and its receptor (bottom)

Ferritin capsule - bottom cut in half to show chamber that can contain ~4500 iron ions

IRE-binding protein (IRE-BP). And when it’s bound to that protein it prevents translation…of the mRNA. But iron will cause the IRE-BP to dissociate from the iron response element, and then translation can occur. So if there is no iron, then you’re not making ferritin, because you don’t need it - you don’t have any iron to store. But if iron starts showing up, then you start making ferritin, and then you can start storing the iron the way that you’re supposed to.

Figure 40.21 The iron-response element. Ferritin mRNA includes a stem-loop structure, termed an iron-response element (IRE), in its 5ʹ untranslated region. The IRE binds a specific protein that blocks the translation of this mRNA under low-iron conditions.

Here on the left we have the IRE, just a simple stem-loop with a couple of little bases sticking out, but it forms a recognizable structure that the IRE-BP can bind to.

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Diagram of the Iron-Response Element

• Ferritin mRNA contains a stem-loop structure in its 5’ untranslated region called the iron- response element (IRE). In the absence of iron, the IRE-binding protein (IRE-BP) binds to the IRE and prevents translation. When present, iron binds the IRE-BP, causing it to dissociate from the IRE, thereby allowing translation occur.

Figure 40.22 Transferrin-receptor mRNA. This mRNA has a set of iron-response elements (IREs) in its 3ʹ untranslated region. The binding of the IRE-binding protein to these elements stabilizes the mRNA but does not interfere with translation.

Well, as often is the case, nature has figured out a clever way to regulate the production of these proteins, and so this method is reused in the case of the transferrin receptor. So there are several IREs in the 3’ untranslated region of the transferrin receptor, and those will also bind to the IRE-BP - but in this case it doesn’t prevent the translation, it actually stabilizes the mRNA so that it can be translated more! If there’s not enough iron in the cell, if there’s very little, then you want to increase the transferrin receptor, so that you can get more iron. When you end up getting more iron, then the binding proteins can bind to that iron, and they become released from the receptor mRNA, and the receptor mRNA can then be degraded, effectively turning off the signal, and not making anymore transferrin receptor protein.

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The Stability of Messenger RNA Also Can Be Regulated

• Transferrin-receptor mRNA also has several IRE regions located in the 3’ untranslated region.

• When little iron is present, IRE-BP binds to the IRE, allowing the mRNA to be translated.

• When present, iron binds to the IRE-BP, causing it to dissociate from the transferrin-receptor mRNA. Devoid of the IRE-BP, the receptor mRNA is degraded.

Now, here’s a picture of the mRNA for the transferrin receptor, and you can see the coding region on the left is unimpinged - unaffected by these iron-response elements over there on the right. What the IREs do, is when they’re bound with the binding protein, that prevents the degradation of the mRNA. I believe that mRNAs are degraded from the 3’ end. It’s generally easier to chew away nucleotides from a nucleic acid from the end than it is to cut them in the middle, using an exonuclease from the end, rather than an endonuclease from the middle. And that’s another reason, actually, that some people think that using the poly A tail is important is because it protects the mRNA. When it gets chewed away, the poly A tail doesn’t code for anything, so that’s kind of a buffer zone that prevents the important parts of the mRNA from getting degraded. But I’m not entirely sure about all of these mechanisms, so if somebody wants to look into this one a little bit more closely, and report back to me, I will give you extra credit for that.

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Diagram of the Transferrin-Receptor mRNA

Now we’re going to talk about a different mechanism for regulating the translation of mRNA, and that’s called RNA interference (RNAi). And this is an incredibly important mechanism that is not only very common in the body, but is also a new method that is being used for controlling gene expression therapeutically. One thing that happens sometimes is that a virus will get into the cell - and viruses often have double- stranded RNA genomes. There’s not supposed to be these double-stranded RNA genomes, so the eukaryotic cells have a mechanism that basically just goes around and destroys all double-stranded RNA. There’s a protein called dicer which cleaves double-stranded RNA into small fragments, wherever it finds it, and basically “dices” it. These small fragments of RNA are then bound to a class of proteins called Argonaute (that is a single strand of that viral RNA), and then the Argonaute protein will look for complementary strands to that little bit, and destroy them.

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Small RNAs Can Regulate mRNA Stability and Use (1/2) • RNA interference (RNAi) leads to mRNA degradation induced by the presence

of double-stranded RNA, which may be present during certain viral infections.

• Dicer, a ribonuclease, cleaves double-stranded RNA into small fragments. Single-stranded components of the cleavage products, called small interfering RNA (siRNA), are bound by a class of proteins called Argonaute to form the RNA-induced silencing complex (RISC).

• The complex locates mRNA complementary to the siRNA and degrades the mRNA.

http://courses.biology.utah.edu/bastiani/3230/DB%20Lecture/Lectures/WormRNAi.html

Figure 40.23 MicroRNA action. MicroRNAs bind to members of the Argonaute family where they serve to target specific mRNA molecules for cleavage.

You can see here, up at the top of this diagram, we have an Argonaute protein in the center, and it’s got a red RNA that is complementary to the white RNA. And wherever it finds the complementary sequence to the red RNA, it will cleave it up and therefore prevent it from being translated. Now, this is useful, of course, in the case of dealing with viral infection. Your body can recognize a viral infection and attack the RNA to prevent the virus from making the proteins that it needs. But there is another application - this same system is used to regulate the expression of mRNAs that are made by our own cells. So - you remember how Dicer cuts the double stranded mRNA into small bits? Well, sometimes you can just make a small bit of RNA and introduce it into the system, and Argonaute will grab it and then start cleaving complementary sequences. So our cells actually make these small bits of RNA - they’re called microRNAs (miRNA). And they combine with Argonaute, and once they combine with Argonaute they go around and degrade our own RNA. So it’s a way of turning the RNA off. So by causing the expression of one of these miRNAs, you’re actually shutting down the expression of a different RNA. Once the complementary sequence is found, it can either just sit there and block translation of the mRNA, or it can actually cut the mRNA and degrade it. Random RNA sitting around in the cell is just degraded - that’s a common process that’s always happening - so RNA has a very short life span in the cell, unless it’s protected in certain ways by being bound to

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Small RNAs Can Regulate mRNA Stability and Use (2/2)

• Small RNAs, called microRNAs (miRNAs), are generated from large precursor RNAs encoded in the genome. These miRNAs associate with Argonaute to form a complex that regulates translation in one of two ways. If the siRNA binds to mRNA by precise Watson–Crick base-pairing, the mRNA is degraded. If the base-pairing is not precise, translation of the mRNA is inhibited.

• An estimated 60% of human genes are regulated by miRNA.

other proteins.

Now, you might think this is just something that’s not that important - an interesting thing that happens - but, as it turns out, 60% of our genes are regulated by miRNA. So that is 60%! And this is another layer of regulation. So microRNA, I guess they’re not genes, because they are not translated into a protein, but they might be genes in the sense that they are important components of the cell that get transcribed, and have transcription factors, and are regulated so these miRNAs are only produced when they should be produced. And the great mix of these miRNAs, and your mRNAs, the Argonaute proteins, and all of these things together combine to somehow to make the correct amount of protein for the survival of your cell. And I just find that amazing! So we have several mechanisms for regulating gene expression. Remember - we have our transcription factors, we’ve got the histone code, we’ve got things that regulate the transcription, or the translation, or the editing of RNA, the transport of RNA, the degradation of RNA. It’s an extraordinarily complex amount of regulation that‘s going on - because it’s an extraordinarily complex system. Your cells are unbelievably complex. We’re not even close to understanding most of how it works. We are making good progress, we’re trying, but there is a lot left to be done. So that’s why we need biochemists!

I hope you have enjoyed this course…

…and perhaps you will consider continuing with your biochemistry adventure. And if so I think you should watch this video, because this will show you a little bit of the whimsical side of biochemistry. It was produced by the Stanford University Department of Biochemistry in 1971. And if you are going to be a biochemist, you should be aware of this video, because it’s famous! If you’re not going to be a biochemist, it’s still fun to watch! So congratulations - thanks for participating in the class, and I look forward to hearing any of your questions and feedback. If you enjoyed the class, please write a review - I’m on ratemyprofessors.com and there’s a link at the bottom of the page. Thanks so much and have yourself an interesting life!