Translating the Genetic Code

DANILO V. ROGAYAN JR.Faculty, Department of Natural Sciences

Figure 13.2

An overview of gene expression

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?

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

a

b

c

Any frame is potentially the

reading frame!

OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWa

b

c

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWa

b

c

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

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWa

b

c

ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWa

b

c

^

Reading Frames & Mutations

• 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.

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWa

b

c

ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEWa

b

c

^

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

• 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 …

Possible

Codons

Percentage in the Random

Polymer

GGG 0.7 x 0.7 x 0.7 = 0.34 = 34%

GGU 0.7 x 0.7 x 0.3 = 0.15 = 15%

GUU 0.7 x 0.3 x 0.3 = 0.06 = 6%

UUU 0.3 x 0.3 x 0.3 = 0.03 = 3%

UGG 0.3 x 0.7 x 0.7 = 0.15 = 15%

UUG 0.3 x 0.3 x 0.7 = 0.06 = 6%

UGU 0.3 x 0.7 x 0.3 = 0.06 = 6%

GUG 0.7 x 0.3 x 0.7 = 0.15 = 15%

Decoding the Genetic Code

Radiolabeled Amino Acid Added

(%) Amino Acid

Incorporated

Glycine (GGG + GGU, UGG, GUG)

49

(34 + 15)

Valine

(GGU, UGG, GUG, GUU, UUG, UGU)

21

(15 + 6)

Tryptophan(UGG, GUG, GGU)

15

Cysteine(UUG, GUU, UGU)

6

Leucine

(UUG, GUU, UGU)

6

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

Lakô hã salamát!Maraming salamat!