Lecture 7 Midterm Exam Notes.docx
Lecture 7 Midterm Exam Notes.docx

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Lecture 7/8 Smart Pages: Ch 2 fig 24; Chapter 11 figs 41, 42, 44, 46, 47, 49, 50 Translation (protein synthesis): Synthesis of proteins in the cytoplasm using the information encoded by an mRNA. (11.1, 11.8) Codons: Sequences of three nucleotides (nucleotide triplets) in mRNAs that specify amino acids. (11.6) Frameshift: see above chapter for it. tRNA: an adaptor and brings proper amino acid to the ribosome Anticodon: A three-nucleotide sequence in each tRNA that functions in the recognition of the complementary mRNA codon. (11.7) wobble base (hypothesis): Crick's proposal that the steric requirement between the anticodon of the tRNA and the codon of the mRNA is flexible at the third position, which allows two codons that differ only at the third position to share the same tRNA during protein synthesis. aminoacyl-tRNA synthetase (AARS): An enzyme that covalently links amino acids to the 3' ends of their cognate tRNA(s). Each amino acid is recognized by a specific aminoacyl-tRNA synthetase. (11.7) Initiation codon: A triple AUG, the site to which the ribosome attaches to the mRNA to assure that the ribosome is in the proper reading frame to correctly read the entire message. (11.8) Shine-Dalgarno sequence: Messenger RNA possess this sequence upstream at the 5' end to the start codon Elongation: the second step in translation after initiation A, P, E sites: 3 sites on the ribosome; A= Aminoacyl or Acceptor, P= Peptidyl, E= Exit. Peptidyl transferase: That portion of the large ribosomal subunit that is responsible for catalyzing peptide bond formation; peptidyl transferase activity resides in the large ribosomal RNA molecule. (11.8) translocation: - A chromosomal aberration that results when all or part of one chromosome becomes attached to another chromosome. (HP12) -(definition #2) The steps in the translation elongation cycle that involves (1) ejection of the uncharged tRNA from the P site and (2) the movement of the ribosome three nucleotides (one codon) along the mRNA in the 3' direction. (11.8) polysome/polyribosome: The complex formed by an mRNA and a number of ribosome in the process of translating that mRNA. (11.8) Lecture 7-8: Translation (Proteins synthesis) Protein synthesis - translation 11-2 figure If everything goes as planned genes will be expressed RNA will be processed, Messenger RNA will be transported to the cytoplasm And will then associate with ribosomes which then take The RNA code and convert it to a protein code to form polypeptide chains The elucidation of the genetic code DNA makes RNA which makes protein There are four nitrogenous bases But we know that there are 20 common amino acids which exist in proteins So it is not possible to have only one nucleotide per amino acid because then we would only be able to produce four amino acids And same for two nucleotides Per amino acid because we would only be able to produce 16 amino acids However three amino acids per Amino acids are possible because we would be able to make 64 amino acids And we only need 20 So the genetic code is probably a triplet code Code overlapping or non-overlapping? An overlapping code looks like each nucleotide would be part of two successive Triplets In partial overlapping code Each triplet is sharing Two of the three nucleotides With an adjacent triplet A non-overlapping code is not overlapping How can you tell? You can tell by Looking at the affect of a single base change mutation in overlapping or partially overlapping code. If a single base change mutation in overlapping or partially overlapping code occurred you would Find that more than one amino acid would be changed in the protein because they are sharing information. Evidence for a non-overlapping code: sickle-cell anemia Sickle cell anemia Due to red blood cells that have a sickle-cell shape And clog blood vessels and are not as strong as other red blood cells. Vernon Ingram used protein sequencing Of peptides from hemoglobin From normal and sickle cell patients He found that there is only one change Of glutamic acid to valine switch Which occurs at one position Which is responsible for the different kind of hemoglobin which causes sickle cell shape Mutations Mutations-insertion or deletion of a single nucleotide: If you have one or two insertions This generates something called a frameshift mutation Which causes a missense protein However if you get three Or multiples of three insertions or deletions this restores the reading frame because the genetic code is a triplet code On this life is a series of nucleotide RNA Of triplet codes which can be decoded into the appropriate amino acids Example of an English language sentence of triplet code and the affects of Each number of mutations in each code on understanding The sentence You see that with one or two insertions you get missense mutations but with three (Or multiples of three) the reading frame gets shifted but still makes sense Elucidating the code required: a new tool How does the ribosome know to take a set of instructions in RNA and Create the correct amino acids to make the protein There are four topics relating to this, each with its own name. 1. a new tool, 2. An organic chemist, 3. Brute force, 4. Brilliance. A new tool: in vitro translation system is when you lyse the cells and take its content and put it in a test tube and ask if they can do something With respect to protein synthesis or translation, the molecular machinery is still there but will it work? So you need a functional test to determine if it will conduct protein synthesis You can tell by using radioactive labeling Radioactively label amino acids and see if they will be incorporated into the protein Time is on the X axis and protein is on the Y axis and when we begin there is no action because we still have not added the amino acids. Once you add the radioactive amino acids we get a little bit of radioactive protein being produced but not very much due to whatever RNA remains in the bacterial extract. Once you add more RNA then you get production of radio active protein taking off with a lot of radioactive amino acid being incorporated. 2. An organic chemist- it became possible Conduct organic synthesis of a polynucleotide chain. -Nürnberg and Matthaei were able to take purified uridine or UTP And were able to condense this into polymers of U, etc -They utilize the first tool of in vitro translation and Gave the polymers individually to the extract and asked the question What kind of polypeptide comes out Of the system So they decoded three of the 64 possible triplets This was quite a slow process 3. Brute force - get rid of the word single on this slide!!! Organic synthesis could not be directed to make Defined triplet sets of information But you could mix different percentages of nucleotides together To get polynucleotide chains That were enriched For one or the other Translation of the random co-polymer: three parts U + one part G It's nucleotides are added at random then first you will be adding more u than g Codon is the set of triplets information Here are the possible codons from mixtures of U and G And the statistics on that So for every 100 phenolalanine you get out you see a pattern of the following: About three times less valine leucine and cysteine and And about 10 times less glycine and tryptophan -So the results Indicate that valine leucine and cysteine are encoded by 2U's and 1G and glycine and tryptophan are encoded by 1U and 2G's. But we still don't know the exact specificity of three nucleotides Brilliance- khorana Found a way to make defined triplet sets of information Nuremberg and leder discovered filter binding assays To determine which amino acid was being brought to the Ribosome as these defined triplets were given The filter binding assay -like a coffee filter -it has pores and allows certain materials to go through Based on How big they are and so ribosomes do not go through the filter. The scientists knew that ribosomes are the site of translation, huge in size and could be retained on filter, and other cell components do not bind but can pass through the filter Nurenberg and leders experiment: They set up 20 cell free extracts, Place in tubes, And label each tube from 1 to 20. There is the same thing in every tube except for one Difference - each tube has All 20 amino acids in it But in each case 19 of them are unlabeled And only one is a labeled amino acid With a different labeled amino acid in each tube. Then they took one Triplet and added the same triplet to all the tubes. Then they filtered all of the tubes What they found was that Only the tube that contained the specific radioactive amino acid Only in that case was the filter radioactive Example: Only in ser was the filter radioactive so then they concluded that the uca codes for serine. -Keep this in mind: 1. the same thing is happening in each Tube. The ribosome is there and binds to the messenger RNA. When that happens the appropriate Transfer RNA containing the appropriate amino acids will come and Bind to the ribosome And in every tube the transfer RNA containing serine Is on the filter associating with the ribosome In every case. But only in one case is the Serine radioactively labeled. In each of the other two groups everything is going through the filter Including the radioactive alanine Which was not called for in this particular case -Keep in mind also: 2. There are aminoacyl transfer RNA meaning there is an amino acid attached to the appropriate transfer RNA. These are Adapters that decode The triplets - they come to the ribosome and Bring the appropriate amino acid. If you investigate the specificity of the Triplet uuu. If you would take the tRNAs All of them would pass through the Filter But the ribosomes would not. Is that ribosome has bound to the message for the UUU And the incoming aminoacyl TRNA that encodes is coming with a radioactive phenylalanine The phenylalanine will bind to the ribosome and will not be able to pass through the filter. Which is why only one of the tubes will yield a radioactive filter For each triplet that you give the system Figure 11 – 41 the codon people Do not memorize the fig 11-41 but do learn the trends! Color block suggests similarity If the first is g and second is u then the third doesnt matter cuz they all encode valine 1. So the first two nucleotide Of the codon Specify what the Amino acid is going to be 2. There is only one codon which encodes the Start codon AUG And all protein chains begin with The amino acid methionine which is encoded only by AUG 3. Stop codons are uaa uag and ug
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