Chapter 15 – The Genetic Code and Translation

Beadle and Tatum Neurospora crassa Haploid orange bread mold Haploid? Prototrophs – grow on minimal media; auxotrophs can only grow on media that has been supplemented with substance that it can’t synthesize Irradiated spores to induce mutations

Beadle and Tatum Irradiated spores first grown on complete media, then transferred to minimal media Showed strains that had acquired mutations Series of tubes with minimal media PLUS one additional ingredient If strain grew, indicated mutation affected production of that substance

Sib and Horowitz Biochemical pathway for arginine production I – grew in presence of ornithine, citrulline, arginine II – grew in presence of citrulline and arginine III – grew in presence of arginine Which type codes for which enzyme?

One gene/One enzyme Each gene codes for one enzyme Many proteins are composed of multiple polypeptides, each coded for by a different gene One gene/one polypeptide

Protein Structure Polymer of 20 amino acids Amino acid structure Central carbon atom Hydrogen atom Amino group Carboxyl group R/radical group What defines one amino acid Each R group has different sizes/properties

Amino acid linkage Two amino acids covalently linked together by process of dehydration synthesis Formed bond = peptide bond Long chain called a polypeptide

Levels of Protein Structure Primary structure Amino acid sequence Secondary structure Hydrogen bonds form alpha helices or beta sheets

Levels of Protein Structure Tertiary structure Interactions between R groups Quaternary structure Only present in proteins composed of multiple polypeptide chains How polypeptide subunits are associated to make a complete protein

Genetic code Nucleotide sequence must code for specific amino acids Francis Crick – 3 nucleotides code for one amino acid codon 64 codons 61 code for an amino acid “degenerate” More than one codon can code for the same amino acid

Genetic code tRNA Wobble 30-50 tRNA for 20 amino acids Isoaccepting tRNA have different anticodons but still carry the same amino acid Wobble 1st nucleotide in anticodon pairs with the 3rd codon of mRNA Flexibility in bonding allows one tRNA to recognize more than one codon Still codes for same amino acid

Reading frame Determined by the START/initiation codon AUG – also codes for methionine No overlapping or skipping of bases Termination/stop codons Also called nonsense codons Universality With rare exception, genetic code is read the same by all organisms

tRNA charging Attachment of appropriate amino acid Aminoacyl-tRNA synthetase (20 different) Recognize specific sequences in certain regions of tRNA, and binds the appropriate amino acid to 3′ acceptor arm of tRNA Forms aminoacyl-tRNA

Translation Occurs at ribosomes Attaches to 5′ end of mRNA and moves toward 3′ end Binding determined by Shine-Dalgarno sequence in prokaryotic cells/ modifications in eukaryotic cells

Initiation of translation Ribosome has two subunits – small (30S) and large (50S) Complete ribosome (70S) Initiation factors bind to small unit, prohibiting small unit from binding with large subunit Now free to bind to mRNA Aminoacyl-tRNAmet attaches to initiation/ start codon Large ribosomal subunit attaches

Ribosome Has three sites for tRNA A (aminoacyl) site P (peptidyl) site E (exit) site Initiator tRNAmet enters P site; all other tRNA first enter the A site A→P→E

Elongation Next codon is exposed in the A site Charged tRNA enters A Peptide bond forms between the P site amino acid and A site amino acid Peptidyl transferase Translocation ribosome moves down mRNA molecule P site tRNA enters E site A site tRNA with growing polypeptide enters P site A site now has next codon exposed; ready for next tRNA During next shift, E site tRNA is released into cytoplasm to be re-charged with another specific amino acid

Termination A STOP codon enters A site Release factors enter A site No appropriate tRNA Release factors enter A site Ribosome subunits dissociate Polypeptide is released from last tRNA

Post-translation modifications Methionine cleaved off; chain possibly cleaved Carbohydrates attached (forms glycoproteins) Folding into proper 3D shape Aided by chaperone proteins