Protein Synthesis: Ch 17 From : Kevin Brown – University of Florida Translation Protein Synthesis: Ch 17 From : Kevin Brown – University of Florida

Protein Synthesis Translation from mRNA Codons to amino acids http://www.youtube.com/watch?v=suN -sV0cT6c

mRNA transcript organization 1) Open reading frame: codes for protein 5’end has translational start site, 3’ end has translational stop site 2) 5’ UTR – untranslated region (upstream of start site) 3) 3’ UTR – untranslated region (downstream of stop site)

Typical mRNA molecule structure

Codons Codons are read from 5’ – 3’ in triplets Genetic code 61 codons for amino acids, 20 amino acids Amino acid sequence is determined by the complementary base pairing between mRNA codons and tRNA anticodons

Translation – tRNA molecule tRNA – Transfer RNA – transfers amino acids from cytoplasm to ribosome, where the amino acids get added onto a growing polypeptide chain. Anticodon – triplet code found on tRNA, that is complementary to a specific mRNA codon. Made of a single RNA strand that folds back on itself to form a 3 D structure – L-shaped.

tRNA 64 codons 45 tRNA Why? Last base on tRNA anticodon is flexible – so some tRNA anticodons can recognize more than one codon

Wobble Non standard base pairing at 3’ end of codon – flexibility Many tRNAs have inosine base at 5’ end of anticodon- Inosine can pair with A, U, or G

tRNA bind to specific amino acid Aminoacyl-tRNA synthetase (enzyme) Amino acid P Adenosine ATP i tRNA AMP Aminoacyl tRNA (“charged tRNA”) Aminoacyl tRNA synthetase joins a particular amino acid to a tRNA through covalent bonding There are 20 of these enzymes

Start signals Translational start signal 5’ AUG 3’ , which codes for Methionine Which AUG is the start signal? Bacteria – often have Shine-Delgarno sequence upstream from AUG

Eukaryotes – no Shine Delagarno, but a loose sequence – Kozak sequence

rRNA - ribosomal RNA Ribosome is made up of rRNA & proteins Ribosome – the site of protein synthesis Eukaryotes: Large subunit – 60 S, Small subunit – 40 S Bacteria: Large subunit – 70S , Small subunit – 30 S

Ribosome Schematic Exit tunnel A site (Aminoacyl- tRNA binding site) Small subunit Large subunit P A P site (Peptidyl-tRNA binding site) mRNA binding site E site (Exit site) E

Protein Synthesis - Initiation Initiator tRNA mRNA 5 3 Start codon mRNA binding site Translation initiation complex U A G C P P site i  GTP GDP Met Large ribosomal subunit E

Amino end mRNA E 5 Codons 3 tRNA Growing polypeptide Next amino acid to be added to polypeptide chain

Protein synthesis - Elongation Amino end of polypeptide mRNA 5 E A site 3 GTP GDP  P i A Ribosome ready for next aminoacyl tRNA P site 1) Codon recognition 3) Translocation 2) Peptide bond formation

Protein synthesis - Termination Release factor Stop codon (UAG, UAA, or UGA) 3 5 Free polypeptide 2 GTP GDP  i P

Translation animation http://www.youtube.com/watch?v=Ikq9Ac BcohA

Energy usage during Translation tRNA Charging – ATP to AMP + PPi for each amino acid covalently bonded to tRNA Initiation – 1 ATP to ADP + Pi and 1 GTP to GDP + Pi hydrolyzed during formation of initiation complex Elongation – 2 GTP hydrolyzed per amino acid 1 for codon recognition 1 for energy for translocation of tRNA from A to P Termination – 1 GTP to GDP + Pi hydrolyzed in termination

Completing the protein Folding (may be helped w/chaperone protein) Post-translational modifications: Chemical modifications – add groups Remove amino acids from leading end of polypeptide chain Cleavage of protein

Some polypeptides get targeted to specific locations All ribosomes start free If polypeptide gives signal, ribosome binds to ER. Signal peptide is at leading (NH3) end of polypeptide Signal recognition particle (SRP) – recognized signal peptide and brings into receptor protein in ER Protein either is in ER lumen, or may end up as part of membrane

Mutations Changes in genetic information of cell (or virus) Small scale – few nucleotides: Substitution, insertion, deletion Frameshift are most damaging Large scale – chromosomal changes

Translation http://www.dnalc.org/view/15501- Translation-RNA-to-protein-3D-animation- with-basic-narration.html