Genetic Code Hm

8/14/2019 Genetic Code Hm

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Genetic Code

Presented with Respect toProf. AMRITPAL SINGH

Presented by Patel Hiren M.

M.V.Sc. (Anim.Biotechnology )


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INTRODUCTION

The genetic code consists of 64 triplets of nucleotidescalled as Codons

Each codon encodes for one of the 20 amino acids

Most of the amino acids being encoded by more than onecodon (redundancy)

The genetic code can be expressed as either RNA codonsor DNA codons


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Pathway for Gene Expression


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How do Nucleotides Specify 20 Amino Acids?

4 different nucleotides (A, G, C, U)20 amino acidsPossible codes:

1 letter code 4 AAs 20

A triplet code is the most efficient way to code for all 20amino acids, Shown by Crick et al in 1961


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1. Discovered that frame shift mutations (addition or deletion) resulted in adifferent sequence of amino acids.

20. Also discovered that r+ mutants treated with proflavin could be restored tothe wild type (revertants).

deletion (-) corrects addition (+) or vice versa

Fig. 6.5


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4. Combination of three r+ mutants routinely yielded revertants, unlikeother multiple combinations.

Fig. shows how three nearby + (addition) mutations restore the readingframe, giving normal or near-normal function.


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How was the Genetic Code Deciphered?

1. Cell-free, protein synthesizing machinery isolated from E. coli .(ribosomes, tRNAs, protein factors, radio-labeled amino acids).

Synthetic mRNA containing only one type of base:UUU = Phe, CCC = Pro, AAA = Lys, GGG = ? (unstable)

7. Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA,AAC, AAA) composed of two different bases:

Pro, Lys (already defined) + Asp, Glu, His, & Thr

Proportion (%AC) varied to determine exactly which codon specifiedwhich amino acid .

9. Synthetic polynucleotide of known composition:UCU CUC UCU CUC Ser Leu Ser Leu


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4. Ribosome binding assays of Nirenberg and Leder (1964) (ribosomes,tRNAs charged w/AAs, RNA trinucleotides).

Protein synthesis does not occur. Only one type of charged tRNA will bind to the tri - nucleotide.

mRNA UUU codontRNA AAA (with Phe) anti-codon

mRNA UCU codontRNA AGU (with Ser) anti-codon

mRNA CUC codontRNA GAG (with Leu) anti-codon

Identified 50 codons using this method.

16. Combination of different methods eventually identified 61 codons, theother 3 do not specify amino acids (stop-codons ).


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Triplet Binding Technique

1. Ability to make 3 base length RNAs in specificsequence

2. 3 base length RNA could bind to ribosome

3. Synthesized short RNAs and added them to systemcontaining ribosomes, tRNAs, amino acids, etc.

4. Nitrocellulose filter paper: key ingredient becauseribosomes adhere to paper


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Triplet Binding Technique

5. Mix RNA, ribosomes , etc., and pour through filterpaper

6. Ribosome + mRNA + tRNA (with amino acid) that iscomplementary to mRNA sticks to filter paper

7. Serial experiment: label 1 amino acid each time

8. Assay filter paper for radioactivity


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Cont.

Using these and a few other techniques, the GeneticCode was finally deciphered.

Note: Genetic Code is Degenerate - a specific aminoacid may be specified by more than 1 codon

Note: Genetic code is Unambiguous each codon

specifies (or codes) for 1 and only 1 aminoacid


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THE GENETIC CODE


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Wobble hypothesis

Proposed by Francis Crick in 1966. Occurs at 3 end of codon /5 end of anti- codon.

Result of arrangement of H-bonds of base pairs at the3rd possition.

Degeneracy of the code is such that wobble always

results in translation of the same amino acid. Complete set of codons can be read by fewer than 61

tRNAs.


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Wobble hypothesis


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hypothesis

Pairing in 3rdposition of codonsis Less stringent.

In part, is due toInosine (I), whichcan pair with U, C,or A.Wobble explains

degeneracy of code


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Genetic Code degeneracy

4 different nucleotides (A, G, C, U)

20 amino acids

Wobble position is responsible for degeneracy

18 of 20 amino acids are coded by more than one codon. Metand Trp are the only exceptions

Many amino acids are four-fold degenerate at the third position


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Phenylalani

UUU

Leucin

UUA UU

Serin

UCUUCCUCA UCG

Tyrosin

UA U

S

UAA UAGUGA

Cystein

UGU

Tryptopha

UGG

Leucin

CUUCUCCUA

CUG

Prolin

CCUCCCCCA

CCG

Histidin

CAUCAC

Glutamin

CAA CAG

rginin

CGUCGCCGA

CGG

Isoleucin

AUU AUC

Methioni

AUG

Threonin

ACU ACC ACA ACG

sparagin

AA U

Lysin

AAA AAG

Serin

AGU AGC

rginin

AGG AGA

alin

GUUGUCGUA GUG

lanin

GCUGCCGCA GCG

spartic

GAUGAC

Glutamic

GAA GAG

Glycin

GGUGGCGGA GGG


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Exceptions to the Code

The genetic code is almost universal

Same codons are assigned to the same amino acids and tothe same START and STOP signals in the vast majorityof genes in animals, plants, and microorganisms

However, some exceptions have been found Exa: Mitochondrial genes


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Mitochondrial genes

Mitochondrial mRNA from animals or microorganisms(but not from plants) is placed in a test tube ,cytosolicprotein-synthesizing machinery (amino acids,enzymes, tRNAs, ribosomes) fails to be translated into a

protein

The reason: these mitochondria use UGA to encodetryptophan (Trp) rather than as a chain terminator

When translated by cytosolic machinery, synthesisstops where Trp should have been inserted


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GENETIC CODE: Summary

A. A codon (3 bases) specifies an amino acidB. Sequential and non-overlappingC. Degenerate (more than one codon/amino acid)D. Some codons are start and stop signalsE. The code is nearly universal (see differences in

human mitochondrial code)F. Sequences of bases in genes and amino acids in their

encoded proteins are colinearG. Experiments with synthetic mRNAs established

codon assignments


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Genetic Code Hm

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