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Translating the Genetic Code Gene expression part 3

Translating the Genetic Code Gene expression part 3

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Page 1: Translating the Genetic Code Gene expression part 3

Translating the Genetic Code

Gene expression part 3

Page 2: Translating the Genetic Code Gene expression part 3

Figure 13.2

An overview of gene expression

Page 3: Translating the Genetic Code Gene expression part 3

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?

Page 4: Translating the Genetic Code Gene expression part 3

The Coding Problem

• 1-1 correspondence – NO – 4 possibilities

• 2-1 correspondence – NO – 16 possibilities

• 3-1 correspondence – YES – 64 possibilities

Page 5: Translating the Genetic Code Gene expression part 3

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

Page 6: Translating the Genetic Code Gene expression part 3

Crick’s Experiments to Determine Triplet Nature of Code

Page 7: Translating the Genetic Code Gene expression part 3

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

Page 8: Translating the Genetic Code Gene expression part 3

Any frame is potentially the reading frame!

OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

The actual reading frame is called the Open Reading Frame or ORF

Page 9: Translating the Genetic Code Gene expression part 3

Reading Frames & Mutation Types• Frame shift mutations

– Original reading frame is frame a– Insertions or deletions shift the reading frameROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW

abc

ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEWabc

^

Page 10: Translating the Genetic Code Gene expression part 3

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..

Page 11: Translating the Genetic Code Gene expression part 3

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

Page 12: Translating the Genetic Code Gene expression part 3

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

Page 13: Translating the Genetic Code Gene expression part 3

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

Page 14: Translating the Genetic Code Gene expression part 3

• 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

Page 15: Translating the Genetic Code Gene expression part 3

Polymers of Nucleotide Repeats

Page 16: Translating the Genetic Code Gene expression part 3

Trinucleotide-tRNA Binding Analysis

Page 17: Translating the Genetic Code Gene expression part 3
Page 18: Translating the Genetic Code Gene expression part 3

• 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