Protein synthesis decodes the information in messenger RNA Protein synthesis occurs in three phases: 1. Initiation – the translation machinery locates the start codon in mRNA 2. Elongation – codons are read 5’  3’ as the protein is synthesized from the amino end to the carboxyl end 3. Termination – special proteins hydrolyze the polypeptide from the last tRNA when a termination codon is reached So lets look at protein synthesis, much like transcription and even replication itself it happens in three progressive stages. You need to initiate the process, this takes advantage of a start codon in the messenger RNA so you very precisely put the first amino acid in with respect to the start codon and again because of the triplet nature of the code, that you need to have that exact aligned, every three mRNA nucleotides have to be precisely matched with amino acid. So this sets what we call the reading frame. The start codon is the first three and initiation occurs there and then we continuously add amino acids working 5 prime to 3 prime on the codons and you get elongation to add amino acids constantly to the carboxyl end so that means the protein itself is being synthesized N terminus to C terminus because the are all being added to the C terminal end so you start with the first amino acid and you add to it C terminal end so protein synthesis is N terminal to C terminal and nucleic acid occurs 5 prime to 3 prime and then process needs to be stopped because again there needs to be a very precise protein, there are specific stop signals that end the last amino acid on exactly the right amino acid because that could be a critical amino acid you don’t see any of this sloppy stuff where you see in transcription where you have an extra RNA that can be removed later on or as a no consequence to function. The protein needs to be exactly the right length in exactly the right sequence of amino acids.

Ribosomes have three tRNA-binding sites that bridge the 30S and 50S subunits The mRNA being translated is bound to 30S Each tRNA molecule contacts both 30S and 50S Two of the three tRNAs have anticodon-codon interactions with the mRNA A site = aminoacyl site P site = peptidyl site The E site (exit site) contains the third tRNA tRNA acceptor stems are positioned in 50S So in order to orchestrate this process of bringing in tRNAs and hooking up the amino acids, ribosomes have three critical binding sites for tRNA. Each one with a separate function. The mRNA starts off by binding to the 30S subunits so this basically gets the initiation phase going. The binding of mRNA to 30S and as we will see in a moment that results in the addition of the 50S to create a complete ribosome and the formation of these three sites. The tRNA bridges both of those sites so its reading the mRNA on the 30S subunit and at the same time conducting the business of protein synthesis addition of amino acids on the large subunits so it essentially ties the two together and explains why we need to have the both assembled into a full 70S ribosome. The two sites that are mainly important here are the A site which is the site that’s the acceptor site, that’s why its called that or the aminoacyl site. It brings in the new or accepts the new tRNA with its properly charged amino acid and then the peptidyl site which is where the peptide bond is formed, there is a third site which is called the exit site which is basically the point where the used tRNA is ejected from the ribosome. So the tRNA acceptor stem that is the part holding the amino acid is positioned on the 50S ribosome so the 50S ribosome can very precisely put that amino acid into the peptide. It creates the peptide bond.

So here are those sites. A sequence in a parade, one follows the other So here are those sites. A sequence in a parade, one follows the other. The A site (acceptor aminoacyl site) bringing in the tRNA. The P site which folds the polypeptide chain and is used to add the new amino acid to that polypeptide chain and then the E site which is the spent tRNA which gets ejected.

Here you see the relationship to the mRNA, each of these guys forming codon anticodon interactions to bring the business end of the tRNA molecules into close position so that the peptide that’s on the aminoacyl site can be brought into proximity to the new amino acid being added to it. Then those codon/anticodon interactions are broken in the exit site so that the tRNA can be released.

Playing a movie first then he is coming back to the details Playing a movie first then he is coming back to the details. The links are on the previous powerpoint where I wrote all the links down. Ok so as I mentioned this is a different text books version of that if you are having trouble visualizing that. So we have these three sites and lets go through the steps of initiation, elongation, and termination in detail now.

The start signal is AUG or GUG preceded by several bases that pair with 16S rRNA Nearly half the amino terminal aa residues in proteins from E. coli are methionine, suggesting that AUG is the start codon Initiator regions contain a purine-rich sequence called the Shine-Dalgarno sequence about 10 nucleotides 5’ of the initiator codon The Shine-Dalgarno sequence interacts with a complementary region on the 3’ end of 16S RNA The start signal is the key to initiation, it has a specific codon that binds to a sepcific tRNA in both eukaryotes and prokaryotes a special initiator tRNA is used which is methionine in eukaryotes or formylmethionine in prokaryotes and the businesses help then in prokaryotes only help with a Shine-Dalgarno sequence which is just upstream 5 primes of the initiator AUG codon, so Shine-Delgarno sequence is the consensus sequence that was recognized early in prokaryotic messenger RNA so you just sequenced a bunch of RNAs and they kept seeing this sequence over and over so they thought it must be important for something. It turned out that it formed specific base pairs with ribosomes itself, it helps position the 30S ribosome in the initiation position, so its complimentary interaction with 16S RNA help the 30S ribosome find the start site for translation.

Here we see AUG is always used very precisely to start translation so you get N step or N reading frame translation of the message and that there is some kind of Dalgarno sequence that interacts with 16S RNA upstream to bring the 30S subunit into position.

Bacterial protein synthesis is initiated by formylmethionyl (fmet) tRNA The initiator tRNA (tRNAf) differs from tRNAm used for internal methionine residues The initiator fmet is removed from about half of the proteins found in E. coli from the newly synthesized protein A single aminoacyl-tRNA synthetase links met to both tRNA molecules, however met attached to tRNAf is recognized by a specific enzyme that formylates the met amino group So formylmethionine prokaryotes and methionine in eukaryotes and fmet shows up on about half of proteins that are synthesized in ecoli so when they started looking at the ecoli proteins they realized that fmet probably the initiator amino acid and initiating codon. There is a single aminoacyl-tRNA synthetase that links met to both the tRNA that is used for methionine inside proteins as well as the initiative formylmethionine so tRNA synthetase puts methionine on both but then another enzyme comes along and recognizes the initiator tRNA and formylates, uses formyltetrahydrafolate to create a methyl group on there to create fmet.

So there is our formyl group on there So there is our formyl group on there. So this is done after the tRNA gets loaded with methionine

It is done by this transformylase enzyme It is done by this transformylase enzyme. So the synthetase is the same for both the fmet as well as the met tRNA they both end up with methionine on them but only the methionine bound to the tRNA-fmet gets formylated, transformylase puts the formyl group on and we now have fmet-tRNA. This is happening by a speical enzyme that converts methionine to formylmethionine only when it is bound to the initiator tRNA.

fMet-tRNAf is placed in the P site during formation of the 70S initiation complex Initiation factors IF1 and IF3 join the 30S subunit to preventing 30S from prematurely binding 50S IF2, a GTPase, binds GTP to change shape and enable binding of IF2 to fMet-tRNAf. The IF2-GTP- fMet-tRNAf complex binds mRNA bound to 16S rRNA via theShine-Dalgarno sequence to create the 30S initiation complex Binding of 50S causes GTP hydrolysis, IF’s are released and the 70S initiation complex is formed So now that you have this special initiator tRNA complex it can bind to the 30S subunit and create what we call a 30S initiation complex. As you might guess there are proteins that are involved in facilitating this process that are called the initiation factors so IF1 and IF3 are responsible for binding the 30S subunit and stabilizing it and keeping it as a pre-subunit essentially keeping 30S from binding to make a ribosome and then IF2 is a GTPase that binds to fmet-tRNA and brings it into place with the help of the shine-Dalgarno sequence to put it at the AUG start codon. This is what creates that 30S initiation complex when you got mRNA and you now have it charged with the initiator complex with the first amino acid, you have initiation complex. Then we have the assembly of a functional ribosome by the addition of 50S that comes in, initiaties GTP hydrolysis on the GTP binding protein IF2 and allows the 70S initiation complex to form. IF1 and IF3 fall off, they have done their job of keeping them separate until you get the proper loading of the initiator complex.

So it kind of looks like this So it kind of looks like this. You have a 30S ribosomal unit, it is bound to initiator factors to keep it stabilized as such (IF1 and IF3) until IF2 comes in (the second proteins that loads the initiator tRNA and only in the presence of mRNA) So you have the 30S initiation complex and IF3 and IF1 can leave. GTP is hydrolyzed and now we have got the 50S subunit in association with a 30S so we have a functional ribosome and IF2 is released so the initiation factors come in and do their thing. IF1 and IF3 to keep the structure open and permit mRNA and IF2 to join and then IF2 to properly assemble the 50S subunit to get a functional ribosome. We call this the 70S initiation complex.

Elongation factors deliver aminoacyl-tRNA to the ribosome The mRNA codon in the A site defines which aminoacyl-tRNA will enter the site Elongation Factor Tu (EF-Tu) delivers the correct aminoacyl-tRNA to the A site when GTP is bound Elongation factors as opposed to initiation factors. EF’s instead of IF’s. Then participate in bringing in the individual aminoacyl-tRNAs in to get assembled into the protein. So EF-Tu binds to aminoacyl-tRNAs, it binds to GTP and then assembles those guys into the A site. The initiator complex is currently sitting in the P site

EF-Tu serves two functions: 1. EF-Tu protects the ester linkage in aminoacyl- tRNA from hydrolysis 2. The GTP in EF-Tu is hydrolyzed to GDP only when an appropriate complex between the EF-Tu-aminoacyl-tRNA complex and the ribosome has formed EF-Tu is then reset to its GTP form by EF-Ts which induces dissociation of GDP from EF-Tu and replacement by GTP EF-Tu is involved in allowing the aminoacyl-tRNA to properly get situated in the ribosome so when it forms its bond it will only do so with the peptide that is being grown. These aminoacyl bonds are very labile and reactive and if it weren’t protected it probably would just hydrolyze, water would react with it or it might react with some other group and form an adduct that wasn’t appropriate. So the main function of EF-Tu is to kind of enclose that labile bond until it is in the right position on the ribosome and then expose it right near the C terminus of the peptide that is growing, so that a peptide bond will form and not some other interaction. When that position is found the GTP is hydrolyzed and this allows a conformational change that permits the formation of a peptide bond. The EF-Tu, it now has GDP bound to it so this needs to be recycled so there is another elongation factor called EF-Ts that dissaociates the GDP and replaces it with GTP to recycle EF-Tu in its active form so it can bring in another aminoacyl-tRNA complex.

Peptidyl transferase catalyzes peptide bond formation When both the A and P sites are occupied by aminoacyl-tRNA, the formylmethionine linked to initiator tRNA is transferred to the amino group in the A site. The peptidyl transferase center on the 23S subunit of the 50S subunit catalyzes formation of the peptide bond Formation of a peptide bond is followed by GTP-driven translocation of tRNAs and mRNA Ok so we got everything positioned just right, there is another enzyme activity that forms the peptide bond, that is peptidyl transferase, it combines the group on the AMP site to move the amino acids on the P site to the N terminus of the amino acid on the A site and you have now grown the chain by one strand and of course the peptide is now bound to the tRNA in the A site and that is very transient. Now the ribosome will move to put them back in the P site. This is a GTP driven transloaction, so another GTP hydrolysis elongation factor that binds, called elongation factor G, comes in and translocates the whole mess.

So here we see the peptide sitting in the P site and in coming aminoacyl-tRNA in the A site. Peptide bond formation where the peptdie was on the P site is transferred to the A site and now we need to translocate the ribosome to shift the peptide back to the P site. The tRNA with a peptide back to the P site so this is elongation factor G, which burns GTP in order to move the ribosome with respect to the message bringing what was in the A site now to the P site and opening up the A site for a new incoming aminoacyl-tRNA. So this is what we call translocation, moving the ribosome down one codon. Its shifting the three sites down one codon.

The translocation mechanism This is how EF-G works, it’s a GTPase, basically it takes the site, it gets itself into the A site and does a conformational change to leverage the ribosome along the message, push it down one triplet codon. It does so by hydrolyzing GTP, of course is going to have to be recharged just like EF-Tu. The translocation mechanism

Proteins are synthesized by the successive Ok and so that’s it. I mean then you have the elongation phase is a sequential addition of aminoacyl-tRNAs until you get a peptide that reaches the stop codon and then you have to have termination. Notice the addition is always to the carboxyl terminus, kind of an inside out synthesis where the amino acid is added to the N inside bond always bound by the C terminus. Proteins are synthesized by the successive addition of amino acids to the carboxyl terminus

Protein synthesis is terminated by release factors that read stop codons Stop codons (UAA, UGA or UAG) are read by protein release factors FR1 recognizes UAA or UAG and RF2 recognizes UAA or UGA RF3 is a GTPase that mediates interactions between RF1 or RF2 and the ribosome RF1 and RF2 mimic tRNAs and promote hydrolytic attack on the ester linkage between tRNA and the polypeptide Termination is again inidicaed by stop signals and these guys essentially bind to factors, release factors (RF1 and RF2) that recognize the different stop codons. And when they do so they basically prevent anything else from getting in there. They are the perferred tRNA if you will, the release factor 1 and 3 acts like a tRNA, occupies the A site so no other amino acid can get in there, no other tRNA and then RF3 comes on (it’s a GTPase) that mediates interactions between the release factors creating this artificial tRNA structure. In the absence of a proper aminoacyl-tRNA that is a properly charged amino acid you get that carboxyl group that is bound to the tRNA in the P site reacting with water instead of the incoming aminoacyl-tRNA in the absence of an aminoacyl-tRNA to react with water is preferred and basically hydrolyzes the peptide off and releasing it.

So we see that here, we have an open A site So we see that here, we have an open A site. If this had been a proper codon that would code for an aminoacyl-tRNA a new aminoacyl-tRNA would have come in however it’s a stop codon, there is no good fit for this, RF1 or 3 are a good fit. They come in an occupy temporarily the site and lead to RF2 association and stabilization of the conformational changes in the ribosome itself that favors bonding with water. This bond here(the carboxyl ester linkage to the tRNA is reacted with water instead of the incoming aminoacyl-tRNA) so the peptide is released.

Prokaryotes and eukaryotes differ in the initiation of protein synthesis 1. Eukaryotic ribosomes are larger, consisting of a 60S large subunit and a 40S small subunit. The 60S subunit contains three RNAs: 5S RNA, 28S RNA and 5.8S RNA. The 40S subunit contains an 18S RNA. 2. The initiating amino acid in eukaryotes is methionine rather than N-formylmethionine. A special initiator tRNA is used called tRNAi 3. The initiating codon is always AUG with no Shine-Dalgarno sequence What we have been talking about is there are prokaryotes is a model, 30S and 50S. Eukaryotes are very similar just the ribosomes are a little bit bigger (40S and 60S) and they make an 80S functional ribosome instead of 70S so everything is a little bigger. There is different compositions (he is not going to ask to memorize which RNAs are in which ribosomes, these are secondary issues that aren’t as important as to know that there are significant differences that lead to basically making eukaryotic ribosomes and prokaryotic ribosomes different enough that they make good drug targets. If you drugs that effect prokaryotic ribosomes but not eukaryotic ones then you have an antibiotic) In eukaryotes they don’t use N-formylmethionine they use methionine. In eukaryotes you almost never find methionine as the N terminal amino acid which means that the purpose of methionine in eukaryotes is to start translation and then it gets cleaved off before the protein is used during maturation the protein methionine is removed, which happens only about half the time in prokaryotes. They initiate without shine-dalgarno as far as we can tell there is no shine-dalgarno sequence but instead (next slide)

Use initiation factors that allow small subunits to bind to mRNA with its properly charged initiative acyltRNA and then with ATP hydrolysis, slide the 30S subunit along the mRNA until the initiator anticodon encounters the initiator codon so rather than shine-dalgarno fixing the 30S subunit here we see ATP sliding the 40S subunit until you get that anticodon/codon interaction with the start amino acid.

And so this guy slides along until you get a productive interaction with the 40S subunit and this allows the 60S subunit to assemble and you go from a 40S to a 80S initiation complex. So all parallel to what we see in prokaryotes it’s a little bit different.

Prokaryotes and eukaryotes differ in the initiation of protein synthesis 4. Eukaryotic mRNA is circular. eIF-4E protein binds the 5’ 7-methylguanosine cap and the 3’ poly(A) tail through protein intermediates The messenger RNAs in eukaryotes turn out to be circular because there elongation factors that bind to the 5 prime end and here we see a role for that 5 prime methyl cap. Its needed for elongation factors to bind to and they bring it into association with other proteins that bind to the polyadenylation signal and stabilize the circular message that somehow facilitates translation.

Some antibiotics inhibit proteins synthesis Streptomycin, a highly basic trisaccharide, interferes with binding of formylmethionyl-tRNA to ribosomes preventing initiation Neomycin, kanamycin and gentamycin interfere with interactions between tRNA and the 16S rRNA to inhibit initiation Choramphenicol inhibits peptidyl transferase Erythromycin binds the 50S subunit and blocks translocation So as I was saying, the fact that they are different is a great opportunity for pharmaceutics. Streptomycin for example, is a trisaccharide that interefrers with the binding of the formylmethionyl-tRNA so it blocks synthesis by blocking initiation. Some of these others Neomycin, kanamyin, and gentamycin blcok the interaction between the 16S rRNA and they also block initation in that way. Choramphenicol blocks the peptidyltransferase so even though initiation can occur, elongation can’t occur so you blcok protein synthesis that way. Erythromycin binds to the 50S subunit and inhibits the GTPases so that the thing can’t translocate.

So again beause of their usefulness as drugs this is an area that you want to be familiar with, these drugs inhibit translation and generally how they do it. Puromycin is an analog for aminoacyl-tRNA and so it can get in there, of course it can’t form an elognated chain so it blocks elongation.

Ribosomes bound to the endoplasmic reticulum manufacture secretory and membrane proteins Ribosomes attached to the endoplasmic reticulum to form the Rough Endoplasmic Reticulum (RER) Ribosomes on RER synthesize proteins destined to exit the cell or become membrane proteins exposed on the surface of the cell Three classes: Secretory proteins; lysosomal proteins; and proteins spanning the plasma membrane Ok so that’s the mechanics of protein synthesis which is true for all proteins. I want to end with one other topic which is how you get proteins to leave the cytoplasm and either become membrane proteins or become secreted proteins so they have a slightly different fate and that they end up on ribosomes that become attached to rough ER and you get transported out of the cytoplasm and into the lumen of the ER, and that transport can either be complete in which case they become secreted proteins or it can be partial in which case the protein remains anchored in the membrane of the ER which then fuses with the plasma membrane so you have a membrane protein because it is still anchored in that membrane. So this is how we ended up with secretory proteins, lysosomal proteins that end up in intracellular vesicles and plasma membrane proteins.

So this is all happening on endoplasmic reticulum, the Rough ER in particular because the rough means ribosomes and what they are doing is syntehsizing proteins in the cytoplasm and injecting them into the lumen of the endoplasmic reticulum and basically in doing so they are leaving the cytoplasm and entering the extracellular compartment, they are leaving the cell they are in the extracellular environment that can be realized as a secretory vesicle in which the protein actually becomes extracellular or else it will sit in a lysosome or something like that which is a compartment that is continuous material that is taken from the outside of the cell and the endosomes and you get fusion of those phagolysosomes that now can degrade the protein, but that is all contained well away from the cytoplasm so lets see how that works (next slide)

Protein synthesis begins on ribosomes that are free in the cytoplasm Secretory, lysosomal and membrane proteins begin synthesis on a free ribosome but then arrest until the ribosome binds the cytoplasmic surface of the ER Docking with the ER restarts protein synthesis with the newly synthesized protein threaded into the lumen of the ER Signal sequences mark proteins for translocation across the ER membrane The business here is directing the newly synthesized peptide which is initially cytoplasmic on a cytoplasmic ribosome to the endoplasmic reticulum and their binding sites on the ER that are so called docking protein receptors that allow proteins that bind to the initial part of the newly synthesized protein to take the protein and bring it to the ER so we look for these docking proteins and their receptors to explain that location to the ER. Then the question is what makes the docking protein bind to the new transcript and the answer here is there is certain amino acid sequences which are typically hydrophobic in nature that form signal sequences and when these sequences get made docking protein binds to them.

The translocation machinery consists of four components: 1. The Signal Sequence – 9 to 12 hydrophobic amino acid residues, sometimes with positively charged amino acid residues, usually near the amino terminus of the nascent peptide chain. Some signal sequences are maintained in the mature protein while others are cleaved by a signal peptidase. 2. The Signal Recognition Particle (SRP) recognizes and binds the signal peptide then directs the peptide to the ER lumen. SRPs are GTPases. So signal sequence is short (10 amino acids or so typically hydrophobic) and occasionally depending on the protein some of them will be positively charged it just differs from one to the other. They are amino terminus usually so the first thing synthesized basically determines the fate of the protein whether its going to be secrted or not. IN some cases they are maintained if the signal sequences is maintained it often becomes a membrane protein that is the signal is literally the anchor that anchors this protein in the membrane. In other cases they are cleaved off so the protein can in fact become soluble in the extracellular compartment. So a thing called the signal recognition particle (SRP) recognizes this signal peptide and this is what the docking protein is going to interact with.

The translocation machinery consists of four components: 3. The SRP Receptor binds SRP at the surface of the ER membrane. The SRP Receptor is a GTPase. 4. The Translocon is the translocation machinery that transports the nascent polypeptide across the ER membrane. The SRP receptor binds to that and translocates it as a GPS, so we have a translocon which is a channel through the ER that the peptide actually translocated through.

So lets look at the sequence of events here So lets look at the sequence of events here. We have a newly synthesized protein, here we have the ribosome reading mRNA 5 prime to 3 prime. The amino acid is being assembled N terminal to C terminal so the first thing that pops out on the ribosome is the early N terminus of the newly synthesized protein. If that has a signal sequence in it, it can be recognized by SRP (the signal recognition particle) if it doesn’t have the signal sequence it won’t be and it will become a cytoplasmic protein but if it has signal sequence SRP binds to it, acts as a docking protein to dock the SRP complex to the SRP receptor on the ER. So you end up now anchoring those what were free ribosomes and they become rough ER ribosomes. Then there is a transloaction that is GTP hydrolysis dependent, so GTP is hydrolyzed, translocate the signal peptide through the translocon transport protein in the ER and this basically causes all subsequent amino acids to be threaded through the ER regardless of their sequence so they go through the translocon and end up in the ER lumen and the peptide signal sequence is still anchored to the translocon. Now depending on the fate of that protein, if it’s a secretory protein then signal peptidase will cleave the signal off and that way the protein can be secreted that is it ends up being soluble on the other side of the membrane. If it is a membrane protein the signal peptidase won’t cleave it off (doesn’t have a picture of that) but if you imagine with the signal peptide being left in place then that signal peptide remains embedded in the membrane and that becomes the anchor for the membrane protein. In other cases the whole polypeptide chain is basically one signal sequence after another. In other words it is one hydrophobic alpha helix after another and so the signal sequence injects the proteins in the membrane and then as the ribosomes sequences more hydrophobic regions it basically just folds it into the membrane and it becomes one of those multispan membrane proteins we talked about earlier. So those are the other two alternatives. The question is how does this become a secretory protein? I didn’t explain it that well. What happened here is a protein that was cytoplasmic has been translocated inot the lumen of the ER. Remember what happesn in the ER proteins get destined for export they have essentially left the cytoplasm and now that they are in the lumen of the ER they can form vesicles that separate from the ER and fuse with the Golgi apparatus. In the Golgi apparatus this protein will become highly modified (the business of the Golgi apparatus is to add sugar residues and phosphates and do all those post-synthetic modifications, often cleave the protein down) and it will get processed through the Golgi and it enters the cis stack, that is the closes to the nucleus and processes progress it through the Golgi to the trans face of the Golgi and then vesicles form off the Golgi. These vesicles are secretory vesicles. They contain little packages of this protein after it has been processed through the ER and through the Golgi and now budded off as a secretory vesicle it could also be a lysosome at that point. It’s a vesicle that is made by the same general procedure but it doesn’t get secreted so intracellular organelles like lysosomes and peroxisomes are formed the same way they just don’t get secreted, but if it’s a secretory vesicle it will move to the plasma membrane. That vesicle will fuse to that membrane and dump this stuff outside, its secreted. That is why I talk about this compartment essentially being the outside of the cell because wants in here from a topological point of view its an equivalent of the outside of the cell its just a question of do you have the membrane fusions to get it there. You may want to go back to when early in the course when we talked about membrane structure and the relationship of the ER and the vesicular trafficking.