Genetic Code Hm



Genetic Code 1



Presented with Respect to Prof. AMRITPAL SINGH

Presented by Patel Hiren M. M.V.Sc. (Anim.Biotechnology)

INTRODUCTION 2

The genetic code consists of 64 triplets of nucleotides called as Codons Each codon encodes for one of the 20 amino acids Most of the amino acids being encoded by more than one codon (redundancy) The genetic code can be expressed as either RNA codons or DNA codons

Pathway for Gene Expression 3

How do Nucleotides Specify 20 Amino Acids? 4

4 different nucleotides (A, G, C, U) 20 amino acids Possible codes: 1 letter code 4 AAs <20 2 letter code 4 x 4 = 16 AAs <20 3 letter code 4 x 4 x 4 = 64 AAs >>20 A triplet code is the most efficient way to code for all 20 amino acids, Shown by Crick et al in 1961

Evidence that the genetic code is a triplet code 5

 





1960s: Francis Crick et al. Studied frameshift mutations in bacteriophage T4 (& E. coli), induced by the mutagen proflavin. Proflavin causes the addition or deletion of a base pair in the DNA. Two ways to identify mutant T4:  Growth with E. coli B:  r+(wild type) Turbid plaques  rII (mutant) Clear plaques  Growth with E. coli K12 ( ):  r+ (wild type) Growth  rII (mutant) No growth

1.

Discovered that frame shift mutations (addition or deletion) resulted in a different sequence of amino acids. 6

Fig. 6.5

20. Also discovered that r+ mutants treated with proflavin could be restored to the wild type (revertants). ü deletion (-) corrects addition (+) or vice versa

4.

Combination of three r+ mutants routinely yielded revertants, unlike other multiple combinations. 7

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

How was the Genetic Code Deciphered? 8

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 specified which amino acid.

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

4.

Ribosome binding assays of Nirenberg and Leder (1964) (ribosomes, tRNAs charged w/AAs, RNA trinucleotides). 9 ü Protein synthesis does not occur. ü Only one type of charged tRNA will bind to the tri - nucleotide.

mRNA UUU codon tRNA AAA (with Phe) anti-codon mRNA UCU codon tRNA AGU (with Ser) anti-codon mRNA CUC codon tRNA GAG (with Leu) anti-codon ü Identified 50 codons using this method. 16. Combination of different methods eventually identified 61 codons, the other 3 do not specify amino acids (stop-codons).

Triplet Binding Technique 10

1. Ability to make 3 base length RNA’s in specific sequence 2. 3 base length RNA could bind to ribosome 3. Synthesized short RNA’s and added them to system containing ribosomes, tRNA’s, amino acids, etc. 4. Nitrocellulose filter paper: key ingredient because ribosomes adhere to paper

Triplet Binding Technique 11

5. Mix RNA, ribosomes , etc., and pour through filter paper 6. Ribosome + mRNA + tRNA (with amino acid) that is complementary to mRNA sticks to filter paper 7. Serial experiment: label 1 amino acid each time 8. Assay filter paper for radioactivity

12

13

14

Cont…. 15

Using these and a few other techniques, the Genetic

Code was finally deciphered.

• Note: Genetic Code is Degenerate - a specific amino acid may be specified by more than 1 codon • Note: Genetic code is Unambiguous – each codon specifies (or codes) for 1 and only 1 amino acid

THE GENETIC CODE 16

Characteristics of the genetic code (written as in mRNA, 5’ to 3’): 17

1. Code is triplet. 2. Code is non-overlapping 3. Code is non-Punctuating 4. Code is almost universal. 5. Code is unambiguous 6. Code is degenerate. 7. Code has start and stop signals. 8. Wobble occurs in the tRNA anti-codon

Wobble hypothesis 18

ü Proposed by Francis Crick in 1966. ü Occurs at 3’ end of codon /5’ end of anti- codon. ü Result of arrangement of H-bonds of base pairs at the 3rd possition. ü Degeneracy of the code is such that wobble always results in translation of the same amino acid. ü Complete set of codons can be read by fewer than 61 tRNAs.

Wobble hypothesis 19

Wobble hypothesis Pairing in 3rd position of codons is Less stringent. In part, is due to Inosine (I), which can pair with U, C, or A. Wobble explains degeneracy of code

20

21

22

Genetic Code degeneracy 23

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. Met

and Trp are the only exceptions

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

position

Phenylalani ne UU U UUC Leucin e CUU CUC CUA CUG Isoleucin e AU U AUC AUA Valin e GUU GUC GUA GUG

Leucin e UUA UU G

Prolin e CCU CCC CCA CCG

Histidin e CAU CAC

Methioni ne AUG

Alanin e GCU GCC GCA GCG

Serin e UCU UCC UCA UCG

Tyrosin e UA U 24 UAC Glutamin e CAA CAG

Threonin e ACU ACC ACA ACG

Aspartic Acid GAU GAC

S top UAA UAG UGA

Tryptopha n UGG

Arginin e CGU CGC CGA CGG

Asparagin e AA U AAC Glutamic Acid GAA GAG

Cystein e UG U UGC

Lysin e AAA AAG

Glycin e GGU GGC GGA GGG

Serin e AGU AGC

Arginin e AGG AGA

Exceptions to the Code 25

The genetic code is almost universal Same codons are assigned to the same amino acids and to

the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms

However, some exceptions have been found

ØExa: Mitochondrial genes

Mitochondrial genes 26

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 encode

tryptophan (Trp) rather than as a chain terminator

When

translated by cytosolic machinery, synthesis stops where Trp should have been inserted

27

28

GENETIC CODE: Summary 29

A. A codon (3 bases) specifies an amino acid B. Sequential and non-overlapping C. Degenerate (more than one codon/amino acid) D. Some codons are start and stop signals E. 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 colinear G. Experiments with synthetic mRNAs established codon assignments

30

THANK YOU