Download The Genetic Code

Translating the Genetic Code
Gene expression part 3
An overview of gene expression
Figure 13.2
The Idea of A Code
• 20 amino acids
• 4 nucleotides
• How do nucleic acids
composed of 4 nucleotides
specify the synthesis of
proteins composed of 20
different amino acids?
The Coding Problem
• 1-1 correspondence – NO – 4 possibilities
• 2-1 correspondence – NO – 16 possibilities
• 3-1 correspondence – YES – 64 possibilities
Determination of Triplet Nature of Code
• Aacridines & flavinoids cause
single nucleotide deletions and
insertions respectively
• Insertion of 1 base shifts frame &
creates non-sense mutation
• Deletion of base in mutant
restores frame (suppresses 1st
mutation)
• Recombination between mutants
showed that insertion of 1 or 2
bases or removal of 1 or 2 bases
doesn't restore frame, but
insertion of 3 or removal of 3
allows frame to continue
Crick’s Experiments to Determine
Triplet Nature of Code
So.. The code is 3 letter words, but what about
punctuation?
a
b
c
GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
• There is a message, but one must start at the
right place to read it
• Code written in three letter words - codon
• There are three reading frames, but only one
gives an intelligible message – frame b
– NOW THE CAT SAW THE DOG BUT DID NOT RUN
• A start codon (NOW) and a stop codon (END)
define the frame to use
Any frame is potentially the reading
frame!
a
b
c
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
a
b
c
OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
The actual reading frame is called the Open Reading Frame or ORF
Reading Frames & Mutation Types
• Frame shift mutations
– Original reading frame is frame a
– Insertions or deletions shift the reading frame
a
b
c
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
^
a
b
c
ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
Reading Frames & Mutations
a
b
c
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW
• Once a ribosome begins translation in a particular
frame (a) it does not shift frames
• Therefore, if a mutation shifts the reading frame in the
mRNA, the ribosome will read the wrong frame.
^
a
b
c
ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW
NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W..
Deciphering the Code
• Each amino acid in a protein is specified by 3
nucleotides of codon
• Each codon specifies only ONE amimo acid
• There are 64 possible codons but only 20 amino
acids
• Degeneracy
– An amino acid can be specified by multiple codons
– A given codon still only specifies only one amino acid
Deciphering the Code: Three Approaches
• Synthesis and translation of homopolymer
RNA & ratio polymer RNA followed by
amino acid analysis
• Synthesis and translation of di, tri and tetra
nucleotide repetitive RNA polymers
followed by amino acid analysis
• Triplet RNA-tRNA binding assay and
amino acid analysis
Synthetic RNA Templates and In Vitro Translation
• Synthesis of homopolymeric RNAs
– UUUUUUUUUU, AAAAAAAAAA, CCCCCCCCC, &
GGGGGGGGG
• When translated produced polypeptides
– poly phenylalanine (UUU), polyproline (CCC), polylysine (AAA),
and polyglycine (GGG)
• Therefore 4 codons were determined
–
–
–
–
UUU = phe
CCC = pro
AAA = lys
GGG = gly
Decoding the Genetic Code
• The enzyme polynucleotide phosphorylase
– polymerizes ribonucleoside diphosphates (NDPs) into RNA
– It does not use a template, the order of nucleotides is random
– The nucleotide sequence is controlled by the relative abundance of NDPs
• For example, if 70% GDP and 30% UDP are mixed together, then …
(%) Amino Acid
Incorporated
Possible
Codons
Percentage in the Random
Polymer
GGG
0.7 x 0.7 x 0.7 = 0.34 = 34%
(GGG + GGU, UGG, GUG)
GGU
0.7 x 0.7 x 0.3 = 0.15 = 15%
Valine
GUU
0.7 x 0.3 x 0.3 = 0.06 = 6%
(GGU, UGG, GUG, GUU, UUG, UGU)
21
(15 + 6)
UUU
0.3 x 0.3 x 0.3 = 0.03 = 3%
Tryptophan
15
UGG
0.3 x 0.7 x 0.7 = 0.15 = 15%
UUG
0.3 x 0.3 x 0.7 = 0.06 = 6%
(UUG, GUU, UGU)
UGU
0.3 x 0.7 x 0.3 = 0.06 = 6%
Leucine
GUG
0.7 x 0.3 x 0.7 = 0.15 = 15%
Radiolabeled Amino Acid Added
Glycine
49
(34 + 15)
(UGG, GUG, GGU)
Cysteine
6
6
(UUG, GUU, UGU)
Phenylalanine
(UUU)
3
Polymers of Nucleotide Repeats
Trinucleotide-tRNA Binding Analysis
• Special codons:
– AUG (which specifies methionine) = start codon
• AUG specifies internal methionines also
– UAA, UAG and UGA = termination, or stop, codons
• The code is degenerate
– More than one codon can specify the same amino acid
• For example: GGU, GGC, GGA and GGG all code for lysine
– In most instances, the third base is the degenerate base
• It is sometime referred to as the wobble base
• The code is nearly universal
– Only a few rare exceptions have been noted