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UB BIO 201 - Protein Targeting

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Bio 201 1st Edition Lecture 38Outline of Last Lecture I. What is the code?II. TranslationA. tRNAB. 3 StepsOutline of Current LectureI. WobbleII. Translation SummaryA. Secretory Pathway ExperimentB. Organelle Pathway ExperimentCurrent LectureI. Wobble- Cells tolerate some loose base pairing between the 3’ base of the codon and the 5’ base of the anticodon. (2nd and 3rd base anticodon must match) This is because there are 64 possible codons, 61 amino acids and 3 stop codons, but only 50 tRNAs. GAGcan base with CUC or CUU. ICC anticodon can bind to GGU, GGC, or GGA codon. Mutations do not lead to amino acid identity change when it is mutated in the 3rd base of a codon. II. Translation Summary- 1. Ribosome scans mRNA beginning at the 5’ cap. 2. The ribosome initiates translation at the 1st AUG (start codon). 3. Elongation continues as theribosome translocates 3 based at a time and tRNAs bring in the appropriate amino acids.4. Translation terminates at the first in-frame stop codon (UAA, UAG, UGA)- signals protein to release. -There is a problem though. This newly synthesized protein is in the cytoplasm. mRNA is exported out of the nucleus into the cytoplasm, but translation begins in the cytoplasm. How does the cell get proteins in organelles?These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.A. Secretory Pathway Experiment-Signal Hypothesis- signal in the translated polypeptide ofa secreted protein tells the cell to transport the protein into the RER. The signal is not in the secreted protein. Experiment 1- Purify free ribosomes, then synthesize mRNA for a secreted protein and mix. They engineered mRNA for secreted protein lacking a bit and expressed it in cells. Found that the protein was not secreted. Experiment 2- Purify RER using density gradient centrifugation, then synthesize mRNA for a secreted protein and mix. They added signal peptide to mRNA for a non-secreted protein and expressed it in cells. Found that the protein is secreted. -The signal peptide is a sequence coded for mRNA of many secreted membrane proteins. This is the bit that is lost when protein is translated be RER microsomes. -Co-translational import- For the endomembrane system. 1. Signal peptide binds to signal recognition particle, 2. SRP binds to SRP receptor on the RER, 3. Polypeptide translocates through channel into RER, 4. Signal peptidase cleaves signal peptide, 5. When translation terminates polypeptide release inside channel. Translocation continues in cytoplasm. Hydrophobic stretch is retained in ER membrane. B. Organelle Pathway Experiment- How do cells target proteins not in secretory pathway? -Nuclear localization signal- sequence that targets proteins into nucleus. Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-Experiment- 1. Remove NLS from protein normally found in the nucleus. Inject into cells and observe. 2. Engineer NLS into protein that is normally cytoplasmic. Inject into cells and observe. RESULT: NLS transports protein into nucleus. -Post-translational import- These proteins are transported into their organelles after translation is complete using proteins that recognize the targeting


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UB BIO 201 - Protein Targeting

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