Chapter 15 – The Genetic Code and Translation. Beadle and Tatum Neurospora crassa –Haploid...

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– 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– 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