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BIO240 Lecture 24

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Jennifer Harris

Lecture 12: Translation - Firstly, remember that tRNA brings AAs to the ribosomes. The tRNA is the one that recognizes the mRNA through anticodon to codon pairing. - If you think about this the tRNA already has an AA attached. You have to make sure that you have the right AA attached to the right tRNA otherwise you’re in trouble. What it means is that you need to have 20 different enzymes, 20 different tRNA synthases – each of the 20 tRNAs binds to a particular AA. - Diagram: tRNA that binds to a tryptophan – this particular tRNA synthase only binds to Trp, as well it binds to any transfer RNA that has an anticodon that recognizes the codon for tryptophan. - Recall: for the degeneracy inherent in DNA, there is a subset of tRNA that will have the correct anticodon. In the case of tryptophan there is only one but for others there may be subsets/replacements. - This particular enzyme is binding to the AA and is attaching the AA to the tRNA and it’s happening in the cytoplasm before we get to the ribosome. The enzyme attaches the AA onto the 3’ end of the tRNA (remember tRNA is just RNA). When it’s like this, there is a special nomenclature, when it has AA attached, it is called amino acyl transfer RNA (when it’s got the combo). The reason why is amino acyl side group and tRNA. - As we will see the transfer RNA is going to the ribosome and base pair with the correct codons and you will have the appropriate AA being added. - There are two steps to maintain fidelity – notion that the anticodon must correspond to the correct AA.  The synthase is going to make sure that it’s only binding to the right AA (tryptophan) and any tRNA with the right anticodon.  Another part of fidelity is that in the mRNA, there must be good base pairing – so tRNA anticodon must correctly pair with codon of mRNA. This ensures that you get the correct AA sequence of the protein. - Ex: With Arg, there are 6 different mRNA codons, therefore there are 6 different tRNAs that can fit in there. One synthase will recognize Arg plus the subset of tRNAs (it can recognize all 6 tRNAs with different anticodons). - tRNAs don’t really react with each other since there’s only 3 nucleotides there isn’t that big a patch for combination so the dangers of tRNAs binding together due to complementary base pairing is very small. - What happens with tRNA synthase, it binds to tRNA, adds AA onto the 3’ end of the tRNA. If it adds on the incorrect AA then what happens is the AA will flip into the editing site. If AA were the correct one, then it would not be able to fit into the editing site. Normally the correct AA gets added on but if something goes wrong, then this AA can pop into the editing site of the enzyme and gets chopped off. If AA is added on correctly it cannot fit into the editing site, therefore you don’t get editing – this is only like proofreading – like a check. - This is much like exonuclease activity of DNA polymerase (very similar). The chopping off of the amino acid is called hydrolytic editing – it chops off either the AA or nucleotide. - The ribosome is the action place, where protein synthesis happens. They are found in both eucaryotic cells and prokaryotic cells. There is a lot of RNA in ribosomes. - We see prokaryotic ribosomes on the left, these are found in bacteria and on the right is eucaryotic ribosome. There are two subunits to each ribosome: one large and one small subunit. They have similar kinds of shapes; they both have RNAs and lots of proteins that assemble with the RNAs. - Ribosomes are made of combinations listed in the slide there is an extra RNA in the large subunit in eucaryotic cells. **You don’t need to memorize all the small details just know that there is a lot of RNA in protein in the ribosomes**  Ribosomes are floating freely in cytoplasm/cytosome.  When synthesizing subset of proteins, ribosomes are found on the rough endoplasmic reticulum. - There are 3-4 sites in ribosomes that are important. There is a site that binds to the mRNA, the E, P, and A sites are areas where the tRNA can fit into. Notice you’ve got the large and small subunits – only assembled when they are synthesizing protein, otherwise they are found separate. - We are looking at the process as it is happening so we are in the process of synthesizing protein. - Start from the top left and follow the step numbers. - We already have a small protein growing (small polypeptide chain) from the N terminus. The C terminus is the place that is coming out of the ribosome so if we were to chop it, it would be at the C terminus. - Notice that the little peptide is attached to the P site and it is the tRNA. Because it has a peptide attached it is called a peptidile tRNA. - The elongating chain has already been synthesized and we will witness the addition of the 4 amino acid on. - We have the peptidile tRNA in the P site, a new amino acyl tRNA comes in and binds onto the next codon in the mRNA. If it’s correct, you get a peptide bond formed between 3 and 4 amino acid. The whole ribosome moves along the messenger RNA, it translocates along the mRNA. - Now notice that # 3 is empty and doesn’t have a peptide on anymore and is in the E (exit) site. Now # 4 is in the P site and we will bring in the next AA into the A site. This process is the elongation part of protein synthesis. - Animation: Once an initiator tRNA is positioned at the AUG start codon, elongation of the AA chain proceeds. The next amino acyl tRNA binds the ribosome at A site. The AA at the P site is transferred to the tRNA at the A site. The AAs are linked by the formation of a peptide bond. Peptide bonds are formed by reaction of the alpha amino group of the A site of the AA with the P site of the AA. After peptide linkage, the empty P site tRNA shifts to an exit or E site on the ribosome. The ribosome translocates the length of one codon, releasing the empty tRNA. The A site is free for the next incoming tRNA. The cycle is repeated as the ribosome travels along the mRNA resulting in a growing polypeptide chain. - There are additional proteins that help to make sure that this is going to be an accurate synthesis of proteins. Sometimes you don’t need these proteins but the synthesis is not as accurate and mistakes are made. - The additional factors are called elongation factors – proteins with specific characteristics – they bind to GTP and when they bind to GTP, they have one conformation & when the GTP is hydrolyzed, they have a different shape. This is a common theme you will see as you go through 240 and 241. - Elongation factor TU binds to the amino acyl tRNA and one thing it does is that it ensures that the right AA is binding to the right tRNA – it has the limited ability to make sure that this AA is correct with tRNA. It also escorts this amino acyl tRNA to the right site. - If there is good proper base pairing between the codon and the anticodon, then GTP is hydrolyzed and when the GTP is hydrolyzed, the factor then leaves after changing shape and floats away. There’s proofreading where you must have hydrolysis or else the GTP tRNA complex moves away. Incorrect base pairs move away automatically. EF-Tu brings the right tRNA to the ribosome and ensures correct base pairing. - There is another elongation factor called EF-G and this binds to the ribosome and ensures that the ribosome translocates along the mRNA, it also binds to GTP which hydrolyzes to GDP, once this happens the EF-G moves away - EF-Tu and EF-G are found in prokaryotic cells. Equivalent elongation factors are found in eucaryotic cells, the EF-Tu equivalent is called EF-1 and the EF-G equivalent is called EF-2. These help ensure that synthesis is more accurate and help to move ribosomes along. - In the small subunit of the ribosome, there is the 16S ribosomal RNA which will form base pairs with the codon and anti-codon. - In the figure, it shows only one base and showing base pairing between codon and anticodon and 16S RNA also base pairs & only base-pairs if the correct codon-anticodon pairing is achieved  So in the small subunit of the rRNA there is additional quality control in the sense that the 16S RNA participates in the binding pair. The H-bonding network ensures that binding is accurate. - EF-Tu is shown in combination with a tRNA in grey with red colour and purple. At the top you see the AA in yellow and protein EF-Tu and the GTP binding site (left diagram). - This shows only the structure of the EF-Tu (3 of its domains) and one of the domains binds to GTP (GPPP) – once it hydrolyzes it becomes GDP. Once the hydrolysis occurs, the conformation changes for GDP – the alpha helix of GTP interacts with different domains and GDP has a shifted alpha helix. The top vs. the bottom is a different conformation. The tRNA gets released and the EF-Tu floats away due to the change in conformation. - Elongation factors ensure greater fidelity 1) Yes 2)  Increase speed and efficiency  Ensure error correction mechanism 3) GTP hydrolysis (the switch is mediated by GTP hydrolysis) 4) Binds to the tRNA 5) Helps to move ribosome forward along mRNA - This is showing the rRNA in a ribosome; notice how complex the binding is. The ribosomal RNA takes on huge amounts of double strandedness and forms a good portion of the entire ribosome structure. - Right diagram shows base pairing of different parts of the ribosomal RNA. The 5S and 23S rRNA is seen in 2D. - The interesting thing is that it’s the 23S rRNA that is the one in the large subunit that actually catalyzes the peptide bond formation. - You have the tRNA in the P site with the small protein, the tRNA with an AA which is the amino acyl tRNA in the A site and you have an adenine which is part of the base which is part of the 23S rRNA. It fits in a nice pocket in the 23S rRNA and there is attack of the different bonds – the hydrogen transfers from the amino group, attaches onto the adenine temporarily and then the amino group can attack the P site of carbon which then forms the peptide bond & then the hydrogen gets transferred back onto the oxygen and the tRNA is empty – it no longer has protein. The protein has been transferred onto the AA. There is the empty tRNA in the P site and we see the new petidile tRNA with the amino part and the protein. - The adenine is part of the 23S and it’s the active site where you have the actual catalysis of the peptide bond formation. - To initiate translation you need to get everything assembled. Remember that the large and small subunits aren’t together to begin with – they must be assembled together. - The first step is that the tRNA binds to the P site in the small subunit. The tRNA brings in Met that assembles with the small subunit and to do so, it requires an initiation factor. - Here this is showing what goes on in eucaryotes and that’s why there’s an E. The initiation factor 2 binds to GTP (IF-2 is another switch protein). - The tRNAs are unique, the ones that goes into the first site that will become the P site are initiator tRNAs and always brings in Met. - Then the mRNA with its 5’ cap binds to another couple of different initiation factors & other initiation factors bind to the poly-A tail, they interact together. That then assembles with small subunit and some initiation factors leave. The small subunit moves along until it finds the correct start co
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