Lec 19 - notes PDF

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What is a ribosome and how does it make a protein? And how is translation controlled?! ! Transcription: Location ! • Think about the process of transcription and the difference between prokaryotic and eukaryotic cells. Where would you expect transcription to occur ! ◦ Prokaryotic- Not in the nucleus because there is no nucleus in a prokaryotic cell ◦ Eukaryotic- in the nucleus where DNA is located- happen in the nucleus ! • What does that mean for translation? In prokaryotes transcription and translation can occur nearly simultaneously.. In eukaryotes, transcription and translation can never occur simultaneously because the mRNA has to be exported out of the nucleus so transcription happens in the nucleus, mRNA is made, goes through processing and then the mature mRNA has to leave the nucleus in order to encounter the ribosome and go through translation ◦ In prokaryotes transcription and translation can happen basically simultaneously because there is no separation of location • Where does translation take place in prokaryotes and eukaryotes ! ◦ Prokaryotes- not the nucleus ◦ Eukaryotes- not the nucleus How do ribosomes function in translation?! • Small subunit- holds the mRNA ! • Large submit- where the amino acids get brought together, they get brought together and a bond forms between them at the activation site ! • Activation site- of an enzyme is where a chemical reaction happens, where we have peptide bond formation between the amino acids ! ◦ Amino acids- peptide bonds right between an amino group and a carboxyl group ! • Transfer RNA (tRNA) -an RNA molecule. One end holds the amino acid, the other end has an anticodon- a triplet of ribonucleotides able to form complementary base pairing with the codon for the amino acid in mRNA ! ◦ Aminoacyl tRNA- when a tRNA is linked to its amino acid ! 1. mRNA binds to the small subunit ! A. A sequence in mRNA- ribosome binding site- binds to a complementary sequence in a ribosome ! 2. Anti codon of an aminoacyl tRNA binds to the start Condon in the mRNA! A. First aminoacyl tRNA will always be carrying methionine since the start codon AUG codes for methionine ! 3. The large subunit of the ribosome comes in and binds to the small subunit so that the aminoacyl tRNA is in the middle spot (p)! 4. A new aminoacyl tRNA does into the first (A) spot. Its anticodon binds with the mRNA ! 5. At the active site, a peptide bond forms between the 2 amino acids! 6. Each aminoacyl tRNA moves down one spot so that the one in p is now in the last spot (E) and the one in A is now in P ! A. this cycle continues so a peptide bond forms between the 2 and then it keeps moving down one until a new aminoacyl tRNA comes in ! 7. A new aminoacyl tRNA moves into the A spot and the tRNA in the E spot gets ejected. The cycles continues! 8. Once the ribosome reaches a stop codon. A protein release factor recognizes the stop codon and fills the A site . This causes ! A. the polypeptide and tRNA are released ! B. The ribosome subunits is separate ! Is the ribosome an enzyme or ribozyme?! • Enzyme- protein that catalyzes reaction! • Ribozyme- RNA that catalyzes a chemical reaction ! • Ribosomes contain about an equal amount of protein and RNA- both protein and RNA catalyzes a reaction ! ‣ So how can you categorize ribosome as an enzyme or ribozyme if it contains both protein and RNA?! • Investiture which location on the ribosome would provide you an answer- Active site is where the chemical reaction is happening ! • If the active site is protein- protein is catalyzing the reaction and you could say that the ribosomes •

an enzyme ! If the active site is RNA then its RNA that's catalyzing the reaction and you would call the ribosome a ribozyme ! Turns out the active site is RNA so a ribosome is actually considered a ribozyme- active site is composed of RNA- rRNA- ribosomal RNA •

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Compare: mRNA vs tRNA vs rRNA:! 1. a brain cell and a liver cell would have the same sequence of tRNA and rRNA A. They wouldn't be the same mRNA because they are different cells so different genes are going to be expressed, but they have the same sequence of tRNA and rRNA because these are actually preforming a function of translation a. You need to have the exact same tRNA because it preforms tRNA functions in the same way in every single cell, brings in those amino acids so they have to be structured the same way b. Same thing for rRNA- ribosomes function the same way in all cells so you would need that RNA to be the same in all different cells 2. Translated into a protein mRNA! 3. Required for the process of translation tRNA- brings in the amino acids , rRNA- functions in teh ribosome ! 4. Binding to amino acids tRNA ! 5. Catalyzes the peptide bond between amino acids rRNA ! • Cells have to control which genes get expressed and they need this to respond to changes in the environment and also to have a unique identity- gene expression ! • Transcriptional methods of gene expression control ! ◦ Prokaryotes ! ‣ Operons and how operons function ! ◦ Eukaryotes ! ‣ Level 1 chromatin remodeling (methylation/ acetylation) ! ‣ Level 2 transcription factors and promoters ! ‣ Level 3 mRNA alternative splicing ! ! ! ! ! ! ! Translational methods of gene expression and control:! • After we get that mRNA what are the additional control mechanisms that specifically a eukaryotic cell can do?! • Level 4- RNA interference- when the cell essentially can destroy an mRNA (mature mRNA) so that it never gets translated into a protein ! ◦ Single stranded RNA ( microRNA), works with a protein complex! ◦ Complementary to the mRNA that is the target to be destroyed !

◦ Binds to it and thus preventing RNA from being translated ! miRNA (microRNA) binds to RISC- RNA induced silencing complex! miRNA and RISC bind to a complementary sequence on the mRNA ! A. MicroRNA is going to have a sequence that's complimentary to the mRNA so that they can bind together ! 3. Enzyme embedded in RISC either inhibits the mRNA or destroys the mRNA. Either way, the mRNA is not translated ! • RNA interference happens naturally in the cell and scientists have figures out how to manipulate RNA interference in the lab so that we can knock out a gene! ◦ We can use to stop a gene from being expressed and figure out what the function can be ! ‣ For example, a scientists wants to better understand the effect of a particular gene. She introduces a strand of RNA that is complementary to the target gene mRNA. The target gene will not be expressed so you can compare individuals with the gene expressed vs the ones with it not expressed in order to figure out what function it has 1. 2.

Level 5- folding, cutting and destroying- method fo control after you have an amino acid chain ! ◦ In the end-membrane system, after the primary sequence, right after the primary structure is completed, most proteins need to go through processing. They have to go through the rough ER and the Golgi apparatus to be processed and specializes in other for it to be functional ! ◦ Folding- proteins can fold spontaneously, or the process can be guided by molecular chaperone proteins (in the RER) ! ◦ Chemical modifications- carbs and other molecules get added (usually in the RER) ! ◦ Amino acids removed- sometimes amino aids are chopped off a protein (in the GA)! ‣ This is the reason why even though all amino acids start with methionine, not all proteins start with methionine because sometimes amino acids get removed in this modification process! ◦ Post translational control- during this modification process, the cell can fold incorrectly, chop the amino acid chain in half or destroy it, in order to make the protein nonfunctional... thus controlling gene expression ! • Level 6- inactivation- when a protein gets inhibited ! ◦ after the modification process happens, usually after the protein has even been functional for a while.! ◦ Gets inhibited by phosphate groups, phosphorylation, or enzyme regulator molecules (inhibitory molecules)! • Think about all the levels of gene expression control..! ◦ Which control mechanism is most energy efficient ! ‣ Transcriptional control because you stop gene expression at the level of transcription. You don't waste energy making an mRNA and then making a protein- most energy efficient to just stop gene expression at level of transcription ! ◦ Which control mechanism allows for most rapid response to change in environmental conditions !

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‣ Post translation control all of a sudden you have the environment that changes, going back to transcriptional control mechanism takes time , you still have those proteins that are active in the cell, going back on transcriptional control isn't going to stop those proteins that already exist from functioning. - fastest way to respond to changing environmental conditions is to inhibit those proteins Due to a lack of glucose, a cell has been preforming lipid metabolism for several hours. Suddenly, all the lipid in the environment is eliminated and is replaced with glucose. What is the fastest way that the cell can adjust to this change ! ◦ Inactivate the proteins associated with lipid metabolism- preventing the transcription of proteins associated with low metabolism would be the most energy efficient way, but if the cell needs to respond quickly, inactivated proteins is the way to go

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