BIO 201 Cell Biology with Todd Hennessey Week Fourteenth Notes
BIO 201 Cell Biology with Todd Hennessey Week Fourteenth Notes BIO 201
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This 18 page Class Notes was uploaded by ChiWai Fan on Wednesday May 4, 2016. The Class Notes belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 54 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
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Date Created: 05/04/16
Cell Bio on May 2, 4, 2016 (All images taken from Professor Hennessey’s slide—edited by ChiWai Fan) Cracking the code Hypothesis: an artificial mRNA containing only one repeating base will direct the synthesis of a protein containing only one repeating amino acid. Method: Prepare a bacterial extract containing all the components needed to make proteins except mRNA o Add an artificial mRNA containing only one repeating base Result: The polypeptide produced contains a single amino acid FYI: The Universal Genetic Code is not universal This makes it difficult to express human genes in Paramecium. Instead of stopping at a UAA or UAG, they put in a glutamine and keep going Transfer RNA Not all tRNA are the same Two main parts to tRNA: Amino acid binds to one place and the anticodon at another. That anticodon on tRNA has to match codon on mRNA Anticodon is on tRNA; Codon is on mRNA; codon on mRNA is determined by DNA sequence Transfer RNA (tRNA) contains the anticodon for a specific amino acid What tRNA has to do: 1. It has to be “Charged” by binding its cognate (correct) amino acid 2. It has to bind to the appropriate codon on the mRNA 3. It has to transfer its amino acid to the growing peptide chain 4. It has to move across the ribosome Charging the tRNA There are about 20 different amino acids There are about 20 different tRNAs There are about 20 different amino-acyl tRNA synthetase—their job is to charge up tRNA with appropriate amino acid. Each tRNA reacts with a specific amino-tRNA synthetase to put a specific amino acid on it. Each amino acid has its own tRNA Summary: A specific amino-acyl tRNA synthetase ensures that the right amino acid gets put on the right tRNA Ribosome Structure The first charged tRNA binds to the P site (usually AUG) The second charged tRNA binds to the A site This binding is determined by the complementary sequences on the tRNA (anticodon) and mRNA (codon) You want to put amino acids together in the right sequences because a protein is determined by Amino acid sequence Initiation of Translation 1. The small ribosomal subunit binds to the mRNA 2. Met-tRNA inds to the AUG start codon; 3. The large ribosomal subunit joins to the initiation complex 4. The met-tRNA binds to the P-site Elongation 1. Codon recognition by the next charged tRNA 2. This tRNA binds to the A-site 3. A peptide bond is formed (by peptidyl transferase) between the amino acids, adding an amino acid to the tRNA on the P-site; peptide bond holds amino acids together in protein 4. The free tRNA (with no amino acid on it) moves to the E-site and gets released 5. Another charged tRNA gets added to the A-site 6. Keep repeating until termination Ribosome moves from 5’ to 3’. Termination of Translation Translation ends when a stop codon enters the A-site Stop codons do not bind to any tRNA anticodons Instead, they bind a release factor The nascent peptide is released from the last tRNA and that tRNA is released from the ribosome Everything separates to be used again; recycles Translation on free polysomes Instead of starting with one ribosome, you might have a number of ribosomes (polysomes) on mRNA Each ribosome may have different lengths of protein coming out of it Destinations for Newly Translated Polypeptides in a Eukaryotic Cell Amino acid Localization Sequences direct the proteins to go where they should If no NLS, protein stays in cytoplasm since no signal to go elsewhere If Signal sequence: go to rough ER to get translated by Cotranslational translocation on RER (change in location as you get translated) Cotranslational translocation on RER Synthesis of integral membrane proteins on RER If the nascent protein has a hydrophobic STOP TRANSFER SEQUENCE it will stop further translocation during translation and make it an integral membrane protein; it stops before entering RER. If it doesn’t have a stop-transfer sequence, it will be a soluble protein instead of an integral membrane protein Two amino acid sequences are necessary for the synthesis of an integral membrane protein: Signal sequence (to go to RER) and stop-transfer sequence (don’t go to RER, become integral) Protein folding by a chaperone A chaperone can change the conformation of a protein; comes out with a appropriate conformation If you get messed up, use chaperone to fix it. Posttranslational Modifications of Proteins Proteolysis: make 1 big protein and cut up into smaller ones with different shapes Glycosylation: done to protein after it’s translated Phosphorylation: change conformation and activity and charge of protein Are phosphorylation and glycosylation the only kind of covalent modifications we have heard of so far? Disulfide bonds Ubiquitination—puts on ubiquitin and tags for degradation Acetylation—chromatin remodeling Directed movements of transport vesicles by COPI, COPII and Clathrin Trucks getting into appropriate factory: protein coat helps identify the vesicles‘s origin and destination Clathrin: Moves materials from TGN to lysosomes, endosomes and plant vacuoles. Also from plasma membranes to endosomes; COPI: coat proteins for retrograde movement toward RER; going backwards by coming back to RER COPII: Anterograde away from RER; Why go backwards (retrograde)? Retrieval of RER enzymes that get into Golgi RER proteins that contain the amino acid sequence KDEL (lys, asp,glu,leu) should stay in RER Some RER enzymes accidentally “escape” to Golgi in COPII vesicles These enzymes can be retrieved by KDEL receptors in COPI vesicles and returned to RER Could a lysosomal enzyme be “fooled” to go to RER by adding a KDEL sequence to it? YES Could an RER protein be forced to leave by changing the KDEL to something else? YES, mutation Lysosomal enzyme targeting by Mannose-6-P 1. An enzyme destined for the lysosome is phosphorylated on a mannose sugar in the cis golgi. If the protein has specific mannose 6 phosphate, now the cell is not looking for the protein, it is looking for mannose 6 phosphate; it will go to somewhere else. Where was it made? It goes to RER to become lysosomal enzyme 2. Mannose-6-P receptors in trans golgi bind this protein and packages it into vesicles 3. Clathrin binds to the outside of these vesicles, targeting them to become lysosomes 4. The lysosomal enzyme is released from the receptor as the lysosome matures 5. Clathrin is released and the receptors are recycled to the golgi 6. Some mannose-6-P receptors that ended up on the plasma membrane are retrieved and recycled. How did they get there? If the mannose 6-P receptors are facing inside of vesicles, then vesicles fuse with membrane. An enzyme in ER can be targeted to become lysosomal enzyme by adding a mannose 6-P to it. A mannose 6-P receptor sees that, drags the protein out and puts it on lysosome. All enzymes of lysosome will have mannose 6-P on it. Summary of Protein Targeting Protein trafficking by membrane vesicles What do the COP proteins and Clathrin do? 1. Help to form vesicles by causing membrane curvature and budding 2. Help provide a mechanism for selecting proteins to go into vesicles 3. Help provide a mechanism for vesicle identification so they can go to the right place Concept for Transmembrane Receptor binding 5/4/16 Another way for a vesicle to know where to go V-SNARE: Protein on vesicle T-SNARE: Protein on the target membrane Botulism and tetanus toxins (botox) are specific proteases that destroy SNAREs, preventing neurotransmitter release from motor neurons to muscle Cloning, stem cells, Gene Therapy and creating artificial life Cloning a Mammal Egg cell from sheep #2 (ewe #2) Take the nucleus out of the unfertilized egg cell and discard it (enucleate) Mammary cell from sheep #1, Take nucleus out of mammary cell and put it into the enucleated egg cell; started cell division with electric without sperm involved Put this cell fusion product into a surrogate mother Dolly died after 6 years instead of 12 by natural causes and she had unusually short telomers for a 6 year old Stem cells Each daughter cell can either: A. Go through self-renewal and remain a stem cell and divide indefinitely B. Become terminally differentiated What determines the cell fate (what kind of cell it will become?) Signals for differentiation can be: Genetic (from the cell’s genes) Epigenetic (from other signals) Pluripotent embryonic stem cells ES cell= embryonic stem cells Two Ways to Obtain Pluripotent Stem Cells Theraputic cloning Can we “clone” some of our own cells to replace damaged cells? YES! Should we? Sure why not. A. Embryonic stem cells 1. The early embryo or blastocyst, is cultured in a nutrient medium 2. The outer layer collapses and the inner cell mass are freed from the embryo. Chemicals are added to disaggregate the inner cell mass into smaller clumps 3. Cells grow to a mass of pluripotent cells B. Induced pluripotent stem cells 1. Skin cells are removed from a patient 2. Cell are grown in lab culture 3. A vector carrying several genes controlled by an active promoter is added 4. Cells carrying th vector are selected 5. Cells grow to a mass of pluripotent cells Induced pluripotent stem cells These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by the addition of only a few defined factors Skin transformed into stem cells: human skin cells have been reprogrammed to mimic embryonic stem cells with the potential to become any tissue in the body Gene therapy In vivo and ex vivo Gene Therapy In vivo: put normal gene onto virus and replace good genes to creating infected genes In vitro: replace infected genes with normal genes Viral vectors for gene therapy 1. Some viruses are quite specific for certain cells types, so you can target only certain kinds of cells. 2. Some viruses could be genetically modified to be even more specific or infect different cells 3. The virus can be either a harmless virus (some are) or they could be genetically modified to be harmless 4. If a gene has a mutation that stops production of a protein, the cell could be transformed with the correct gene and the cell would make protein This is a genetic transformation. The “transformation factor” is a normal gene Germline Gene Therapy Genetic curing: if you catch an illness, put the normal DNA in so that it does not have the genetic defect. This is like designer genes (customizing your baby) Creating an artificial cell. Synthetic Life? J. Craig Venter and Daniel Gibson Find the sequence of DNAsynthesize DNAtransform itsynthetic cell The first synthetic cell
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