BIO 344 1st Edition Lecture 15 Outline of Last Lecture I. RNA editinga. Glutamate receptori. NMDA and AMPAb. Modification by ADARc. Other RNA directed modificationsOutline of Current Lecture I. Breaking the genetic codea. Punctuationb. Degeneracy of the codec. tRNAs and the genetic coded. Conservation of the DNA sequencee. Conservation of the protein sequencef. Translationi. Amino acyl tRNA synthetaseCurrent LectureBreaking the Genetic Code- Matching codons to amino acidso Done by Amino acyl tRNA synthetase- 1st break through in reading and establishing the code:o Take poly U, grind bacteria cells, use macromolecules, add in poly U templateo Put radioactive amino acids in each tubeo Allow translation to occur and filter Will only filter polypeptides C14 gets trapped on filter, only for Phe- Poly U codes for poly phenylalanineThese 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.o Also experimented with UCUCUCUCUCUCU… and got Leu and Ser Told them that codons code be 2,3, or 4 nucleotides if codes can overlap Without overlap the code could be 3 nucleotides, or even 5o With UUCUUCUUC they got Phe, poly Ser, and poly Leu This tells you the codon must be 3 nucleotides because there were three possible reading frames that result in those amino acids- UUC, UCU, or CUU- Punctuation—where does coding start and stop, how are codons reado Maybe there’s punctuation after each codon ATGXCATXATTX to code for M, H, I But if there were internal marks to define each codon, frame shift mutations would not existo Relationship between ends of mRNA and ends of protein-- Punctuation on ends of open reading frame Transcription start site is upstream from translation start site Finding the first AUG in the orf is crucial- If started on nt off, all amino acids would change- Also, AUG within orf after the first AUG from the translation start codes for methionine but is not also considered a start codon- Genetic code is degenerateo Some codons code one amino acid per codon, some amino acids are coded for byseveral amino acids With DNA sequence, we know protein sequence With protein sequence we do NOT know DNA sequenceo 4^3=64 possible codons (4 possible nucleotides, 3 per codon)- Genetic code read by tRNAo 3’ acceptor site Where amino acid charges the tRNAo Anticodon 3 nucleotides on the tRNA that basepair with the codon on the mRNA in the active site of the ribosomeo How many tRNAs are necessary? Between number of amino acids and codons-- ~20-64o Multiple tRNA can be used for single amino acids due to wobble= isoacceptors AAA anti codon binds to UUU codon- Will also pair with AAI or AAGo G-U base pairing is allowed in wobble position and I looks like G- UUC codon will NOT pair with AAA anticodon but will with AAI andAAGo A-C base pairing is not allowed in the wobble position but G-C base pairing is, and I looks like G- Conservation of DNA sequenceo Look at the same gene in multiple species to see which nucleotides are conserved within the gene sequenceo The more conserved sequence is typically the exon, because the exon codes for proteino Can identify orfs by looking for variability every 3rd nt (wobble) and by looking for the location of stop codonso How do organisms take advantage of degeneracy? Protection from mutation Gene expression- Can adjust sequence to enhance binding and still get the desired protein- In one to one amino acids (one tRNA per one codon), ribosome will need to wait longer, so expression of these proteins are lower than when multiple tRNAs are capable of bringing the same aminoacid (more efficient, higher expression)- Robustness in genetic code—conservation of protein sequenceo Effect of transition mutation in the third position None for amino acids with complete wobbleo Effect of transition mutation at 1st position Will get a different amino acid, but with the same biochemical properties (such as all polar or nonpolar)- Translationo Ribosome stabilizes tRNA bound to mRNA tRNA anticodon binds to specific mRNA codon that base pairs with the anticodono experimenters learned which codons code for which amino acids by determining which were most stabilized by which trinucleotide mRNAo amino acyl tRNA synthetase= determines the genetic code class I or class II charges the tRNA with amino acids 20 different aa tRNA synthetases- Should recognize all codons for an amino acid- Attach the appropriate amino acid to the 3’ acceptor site if the appropriate tRNA Requires ATP- Activated by ATPdrives coupling Requires recognition of anticodon loop and acceptor stem of tRNA- If aa tRNA synthetase gives tRNA the wrong amino acid, tRNA does not know and the wrong amino acid will be incorporated (tRNA is a passive player)- But aa tRNA synthetases have proofreading activityo Can identify tRNAs charged with the wrong amino acid, bind to tRNA and remove the wrong amino acid so the now uncharged tRNA can be recharged with the proper amino
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