BIO NOTES FOR FINAL 4/23/13 – Translation: From RNA to Protein The Big Picture:- RNA is translated into protein via the genetic code: each amino acid is encoded by a 3-nucleotide combination called a codon- Transfer RNA (tRNA) is the translator for the genetic code, matching the correct amino acid with each codon- The ribosome is a molecular machine that catalyzes peptide bond formation to synthesize proteins in the N-terminal-to-C-terminal direction (amino group to carboxyl group)- Initiation complex – assembly of mRNA, met-RNA, and ribosome- ……….The Genetic Code- 20 amino acid but only 4 nucleotides in each DNA and RNA- Genetic code: Each “word” in the code is a 3 letter combination coding for a single amino acid – the codon- Since a 3 nucleotide code produces 64 different combinations, sooo most amino acids are specified by multiple codons (more than one way to code for 1 amino acid in RNA)- 3 codons (UAA, UAG, UGA) do not specify amino acids, but instead designate the end of the protein sequence: “stop codons”- The 4th special codon is AUG: codes for methionine and also indicates the translational initiation point: “start codon”- The reading frame has 3 possible positions of where the codon begins, and that’s why we need the start codon AUGtRNA: the RNA-Protein Translator- Job of translating mRNA codons into amino acids is done by transfer RNA / tRNA- Each tRNA brings one amino acid to the growing polypeptide chain and then the ribosome will add the new amino acid to the chain by a peptide bond- Ribosome does not add free amino acids in the cell, it must come in in tRNA- tRNA is a short (`~80 nucleotides) RNA molecule that can fold up (bc it can base pair up with itself) into a specific structure with 3 stem-loop regions: “cloverleaf” structure- One loop contains a 3 nucleotide sequence that is complementary to the codon: the anticodon- The amino acid is attached to the 3’ hydroxyl- When folded into 3-D, it has a L shape, with anticodon at one end and amino acid at the other- Each tRNA can carry a single amino acid, but a given amino adic may use several different tRNAs- Anticodon loop at the bottom, hydrogen bonds that make 3D L structure, amino acid attachment site – still antiparallelMatching Amino Acid to Codon- Requires 2 molecular recognition events- 1) correct amino acid must be attached to specific tRNA- 2) correct tRNA must be matched to codon in mRNAo there is some flexibility here: the 3rd position can be flexible and can tolerate a mismatcho this is called wobble pairing and permits U to pair with either A or G, but only at the 3rd position o ex: codon: AAU anticodon: UUGCharging the tRNA- Amino acid is matched and attached to tRNA by the enzyme aminoacyl-tRNA synthetase (requires ATP to work)- Each amino acid has its own aminoacyl-tRNA synthetase, so a given enzyme must recognize every one of the tRNAs associated with an amino acid- Recognition and attachment of amino acid to tRNA requires hydrolysis of ATP to AMP (requires 2 ATP aka 2 phosphate groups) (high energy bond)- Amino acid is first attached to AMP, then transferred to tRNA- tRNA with amino acid attached is called “charged” tRNAThe Ribosome: Molecular Machine- Where the process of matching charged tRNA to codon and catalyzing protein synthesis takes place- Ribosome consists of 2 subunits (large and small) each made up of a large number of proteins plus several ribosomal RNAs / rRNAs- Bacterial and eukaryotic ribosomes are similar in overall structure, but there are significant differences. Some important antibiotics exploit these differences to affect only bacteria____________- Once assembled, the ribosome binds to mRNA, growing peptide chain and tRNAs- Ribosome contains 3 binding sites for tRNAs:o A site holds incoming tRNA with new amino acido P site holds the tRNA attached to growing polypeptideo Discharged tRNA exits thru E site- As protein synthesis occurs, ribosome moves along mRNA, and tRNA, is shifted from A to Pto E site- Growing polypeptide passes thru exit tunnel (on large subunit) where it will eventually be released - Look at pictures in textbook especially the schematic model with mRNA and tRNABuilding a Polypeptide- Just like mRNA transcription, protein translation consists of 3 stages: initiation, elongation, and termination- During initiation, ribosome assembles with mRNA with the tRNA carrying first amino acid (methionine), and several initiation factors- First, small ribosomal subunit binds mRNA plus initiator tRNA – a special tRNA that binds methionine, and is only used for initiation- In bacteria, the small ribosomal subunit can bind mRNA and initaitior tRNA in either order- Small ribosomal subunit binds mRNA at a specia sequence just upstream of the AUG start codon- In eukaryotes, the small ribosomal subunit binds to initiator tRNA first and tehn to 5’ cap of mRNA- For both, the small subunit “scans” (moves from 5’-3’ direction) mRNA until it reaches the first AUG, which binds anticodon CAU of tRNA- Once AUG is reached, the large subunit binds, forming the translation initiation complex- This is facilitated by several initation factors that use energy from GTP (not ATP) hydrolysis to ensure proper assembly- At this point, the initiator tRNA (plus attachment methionine) sit in the P site____Elongation- Elongation is the 1by1 addition of amino acids to the growing chain- Codons in mRNA are read in the 5’-3’ direction, and the polypeptide elongation occurs in N-terminal to C-terminal direction- Bond formation is believed to be catalyzed by rRNA: the ribosome is a ribozyme- Elongation is facilitated by additional proteins called elongation factors- Elongation cycle is a repeated process:o New charged tRNA enters the A site and the codon pairs with the anticodon in that positiono Ribosome “proofreads” match, using GTP hydrolysiso Peptide bond is formed between the C-terminus of the growing chain and amine group of new amino acid – entire peptide chain is transferred from P site to new tRNA at A siteo Ribosome translocates exactly one codon in the 3’ direction moving new tRNA to P site and old tRNA to E site – another GTP is hydrolized hereo Old tRNA is released thru E site and the ribosome is ready for the next aminoacyl tRNA (charged tRNA)- E P and A sites are located on the first 3 codons and during elongation they move one codon from the 5’ end to the 3’ end4/25THE BIG PICTURE:- During elongation tRNAs bring new
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