Translating the Genetic Code Gene expression part 3

Translating the Genetic Code

Gene expression part 3

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

abc

Any frame is potentially the reading frame!

OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

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 frameROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW

abc

ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

^

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.

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEWabc

^

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