chapter 15 – the genetic code and translation. beadle and tatum neurospora crassa –haploid...
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Chapter 15 – The Genetic Code and Translation
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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
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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
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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?
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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
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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
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Amino acid linkage
• Two amino acids covalently linked together by process of dehydration synthesis– Formed bond =
peptide bond
• Long chain called a polypeptide
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Levels of Protein Structure
• Primary structure– Amino acid sequence
• Secondary structure– Hydrogen bonds form
alpha helices or beta sheets
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Termination
• A STOP codon enters A site– No appropriate tRNA
• Release factors enter A site– Ribosome subunits
dissociate– Polypeptide is
released from last tRNA
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Post-translation modifications
• Methionine cleaved off; chain possibly cleaved
• Carbohydrates attached (forms glycoproteins)
• Folding into proper 3D shape– Aided by chaperone proteins