Translation
One nucleotide can’t code for one amino acid, mRNA is read in blocks of three
• 3 nucleotides = codon
• There are 4x4x4 = 64 codons
Which codon codes for which amino acid is determined by genetic code: deciphered in labs
of Mashall Nirenberg, Severo Ochoa & Gobind Khorana; between 1961-1966
How it was deciphered:
Lot of different approaches:
• Possible to isolate an in vitro translation system from E. coli
• When RNA was added to this in vitro system, proteins encoded by RNA were
synthesised
• Polynucleotide phosphorylase was used to synthesise homopolymers of RNA:
o polyU coded for polyphenylalanine
o polyC coded for polyproline
o polyA coded for polylysine
Polynucleotide phosphorylase was used to generate random RNA sequences
Random RNA molecules with defined rations of bases – e.g. a mixture of RNA molecules
containing 76% U and 24% G
• Most common codon in RNA molecules was UUU therefore most common amino
acid in polypeptide chains synthesised was phenylalanine
• Next most common codons will contain 2Us & 1G, next most common amino acids
were valine, leucine & cysteine
• Codons with 1U and 2Gs coded for tryptophan and glycine
DNA of a defined sequence was made chemically used as template for RNA polymerase &
RNA used in in vitro translation – e.g. poly UC message gave (ser-leu)n
Coding binding studies:
• All 64 codons were synthesised
• Analysed which amino acid, joined to its tRNA molecule, was bound to ribosome in
presence of each triplet
• Is a triplet code
• It’s read in 5’ to 3’ direction
• Most amino acids are coded for by more than 1 codon
• Start codon – AUG (met) -gives registering to ribosome
on which triplet to read
• Stop codons are UAA, UAG, UGA
• Genetic code is universal (expect in mitochondria &
chloroplasts both contain small amount of DNA)
• Code is non-overlapping
• Its degenerate because many amino acids have more than one codon
• There’s a 3’ untranslated region = C UAA stop gap
There’re 3 possible reading frames
• Same RNA sequence could, in theory, give rise to 3 different protein sequences
, • Only one of the ‘reading frames’ codes the real protein
sequence
• If insert of delete one base pair of DNA, it changes the
reading frame for the gene & all downstream will be
affected
How codons “read”?
• Amino acids don’t recognise their own codon
• Adaptor molecules are used
• Each adaptor molecule caries an amino acid – covalently bound
• Each adaptor molecule recognises at least 1 codon for a single amino acid – adaptor
will have a complementary codon to codon within the mRNA
• The adaptor molecules are a special class of RNA called transfer RNAs (tRNAs)
• For each codon, there is a tRNA molecule which will recognise it
tRNA – have defined 3D structure
^ this is because there is internal base pairing indicated by the
“clover leaf” structure, also contain modified bases
There are 4 loops:
1. Anticodon
2. DHU
3. TΨC
4. Variable
Like in DNA, base pairing is antiparallel 3’ to 5’, it’s the 3’ end
when amino acid is attached
Amino acid is attached to 3’ end and the anticodon is
complementary to codon on mRNA
The “clover leaf” structure folds to give an L-shaped molecule consisting of 2
helical regions and a bend:
• The two constant loops (DHU & TΨC) form elbow
• The anticodon loop is at one end of the “L” & amino acid is attached
to the other end
All tRNA molecules have a variable loop, and therefore have a slightly
different structure
‘Charging” the tRNA
• A specific enzyme, aminoacyl-tRNA synthetase attaches each
amino acid to each tRNA molecule, thus producing a charged
tRNA molecule
• Variable loop is recognising anticodon, the binding site
specifically recognises a specific amino acid, it covalently
attaches the alanine to the 3’ end of the tRNA – requires energy
in form of ATP, two hydrogen phosphates are consumed
• During synthesis of a charged tRNA one molecule of ATP is
consume, releasing AMP & PPi
• Reaction is driven by the breakdown of PPi to 2Pi
• tRNA carries a lot of energy, will be used to drive formation of peptide bonds
Translation
• The ribosome brings together the amino acid charged tRNA and the mRNA
One nucleotide can’t code for one amino acid, mRNA is read in blocks of three
• 3 nucleotides = codon
• There are 4x4x4 = 64 codons
Which codon codes for which amino acid is determined by genetic code: deciphered in labs
of Mashall Nirenberg, Severo Ochoa & Gobind Khorana; between 1961-1966
How it was deciphered:
Lot of different approaches:
• Possible to isolate an in vitro translation system from E. coli
• When RNA was added to this in vitro system, proteins encoded by RNA were
synthesised
• Polynucleotide phosphorylase was used to synthesise homopolymers of RNA:
o polyU coded for polyphenylalanine
o polyC coded for polyproline
o polyA coded for polylysine
Polynucleotide phosphorylase was used to generate random RNA sequences
Random RNA molecules with defined rations of bases – e.g. a mixture of RNA molecules
containing 76% U and 24% G
• Most common codon in RNA molecules was UUU therefore most common amino
acid in polypeptide chains synthesised was phenylalanine
• Next most common codons will contain 2Us & 1G, next most common amino acids
were valine, leucine & cysteine
• Codons with 1U and 2Gs coded for tryptophan and glycine
DNA of a defined sequence was made chemically used as template for RNA polymerase &
RNA used in in vitro translation – e.g. poly UC message gave (ser-leu)n
Coding binding studies:
• All 64 codons were synthesised
• Analysed which amino acid, joined to its tRNA molecule, was bound to ribosome in
presence of each triplet
• Is a triplet code
• It’s read in 5’ to 3’ direction
• Most amino acids are coded for by more than 1 codon
• Start codon – AUG (met) -gives registering to ribosome
on which triplet to read
• Stop codons are UAA, UAG, UGA
• Genetic code is universal (expect in mitochondria &
chloroplasts both contain small amount of DNA)
• Code is non-overlapping
• Its degenerate because many amino acids have more than one codon
• There’s a 3’ untranslated region = C UAA stop gap
There’re 3 possible reading frames
• Same RNA sequence could, in theory, give rise to 3 different protein sequences
, • Only one of the ‘reading frames’ codes the real protein
sequence
• If insert of delete one base pair of DNA, it changes the
reading frame for the gene & all downstream will be
affected
How codons “read”?
• Amino acids don’t recognise their own codon
• Adaptor molecules are used
• Each adaptor molecule caries an amino acid – covalently bound
• Each adaptor molecule recognises at least 1 codon for a single amino acid – adaptor
will have a complementary codon to codon within the mRNA
• The adaptor molecules are a special class of RNA called transfer RNAs (tRNAs)
• For each codon, there is a tRNA molecule which will recognise it
tRNA – have defined 3D structure
^ this is because there is internal base pairing indicated by the
“clover leaf” structure, also contain modified bases
There are 4 loops:
1. Anticodon
2. DHU
3. TΨC
4. Variable
Like in DNA, base pairing is antiparallel 3’ to 5’, it’s the 3’ end
when amino acid is attached
Amino acid is attached to 3’ end and the anticodon is
complementary to codon on mRNA
The “clover leaf” structure folds to give an L-shaped molecule consisting of 2
helical regions and a bend:
• The two constant loops (DHU & TΨC) form elbow
• The anticodon loop is at one end of the “L” & amino acid is attached
to the other end
All tRNA molecules have a variable loop, and therefore have a slightly
different structure
‘Charging” the tRNA
• A specific enzyme, aminoacyl-tRNA synthetase attaches each
amino acid to each tRNA molecule, thus producing a charged
tRNA molecule
• Variable loop is recognising anticodon, the binding site
specifically recognises a specific amino acid, it covalently
attaches the alanine to the 3’ end of the tRNA – requires energy
in form of ATP, two hydrogen phosphates are consumed
• During synthesis of a charged tRNA one molecule of ATP is
consume, releasing AMP & PPi
• Reaction is driven by the breakdown of PPi to 2Pi
• tRNA carries a lot of energy, will be used to drive formation of peptide bonds
Translation
• The ribosome brings together the amino acid charged tRNA and the mRNA
