Codon - Anti codon Interactions: Wobble Hypothesis

1. CODON-ANTICODON
INTERACTIONS:
WOBBLE HYPOTHESIS.
Shashank Patil
Reg. No. 171233
M.Sc. Biotechnology-1

2. Introduction
 Translation is the process by which the nucleotide sequence of an mRNA is used as a template to join the amino
acids in a polypeptide chain in the correct order.
 It can also be defined as decoding the language of mRNA into polypeptide chain.
 Role of different types of RNA in translation.
1. mRNA: Carries the genetic information in the coded form after replication.
2. tRNA: Plays a major role. Binds to amino acids and carry them to the site of translation.
Helps in growing the polypeptide chain.
3. rRNA: Associates with proteins to form ribosomes. Ribosomes move along the mRNA and
form the growing polypeptide chain.
 Pre-requisite materials to translation: Linking of amino acid to tRNA by aminoacyl-tRNA synthetases. Forming a
high energy ester bond.
 Anti-codon of tRNA molecule interacts with codon of mRNA to add the specific amino acid to the polypeptide
chain.

3. Structure of tRNA
 tRNAs are the folded structures of single stranded RNA.They include
4 bases (Adenine, Guanine, Cytosine and Uracil) and 4 loops (D-loop,
TΨCG loop, Anticodon loop and a shortVariable loop).
 Acceptor stem (7-9 bp long) includes 3` end involved in interaction
with amino acid.The 5` end contains terminal phosphate group.
 CCA sequence present in 3`end is found common in all tRNAs. It is the
site of binding aminoacyl tRNA synthetases.
 The D loop (4-6 bp long) contains a non-standard base,
Dihydrouridine.
 TheTΨCG loop contains Pseudouridine (a modified Uridine), denoted
byΨ.
 Anticodon loop (5 bp long) whose 5`to 3` structure contains 3
anticodon sites binding to 3`to 5` codon sites of mRNA.
 The 1st position of anticodon arm and 3rd position of codon arm are of
wobble in nature. Non standard interaction site.
 The structure of tRNA resembles clover leaf. Hence it is called as
clover leaf model.

4. • Codon: A sequence of three nucleotides which together form a unit of genetic code in a RNA molecule
• Anti-codon: It is also a sequence of three nucleotides forming a unit of genetic code in a transfer RNA molecule.,
corresponding to a complementary codon in messenger RNA.
• Discovery: in 1966, Francis Crick proposed the Wobble Hypothesis.
• Wobble Hypothesis: It is a proof at the molecular level to explain the degeneracy of the genetic code. By this, it is
possible to have non Watson-Crick or non-standard base pairing at the third codon position or the 1st anticodon
position, i.e., the 3` nucleotide of the mRNA codon and the 5` nucleotide of the tRNA anticodon.

5. Standard and Non-standard Base Pairing
Standard base pairing
• Perfect binding of purines to
pyrimidines. A=U and G≡C.
• This type of base pairing occurs in
normal DNA structure.
• The first two positions of codon-
anticodon interaction sites possess
standard base pairing.
Non-standard base pairing
• Altered base pairing. A=T (in mRNA)
and I=C where I is an equivalent base
E.g. Inosine.
• This type of base pairing occurs in
mutations.
• Third position of interaction
possesses the wobble pairing.

6. Inosine
 Inosine (C₁₀H₁₂N₄O₅, Molar mass 268.23 g/mol) is a nucleoside that is formed when
hypoxanthine is attached to a ribose ring via β-N₉-glycosidic bond. It is a basic
material in construction of a cell.
 It is commonly found in tRNAs and is essential for proper translation of the genetic
code in wobble base pairs.
 Adenine is converted into adenosine or Inosine monophosphate (IMP), either of
which in turn is converted into Inosine, which pairs with Adenine, Cytosine and
Uracil.
 Used as a drug to improve athletic performance in 1970`s in East. Uric acid produced
by Inosine has antioxidant property.
 Used in designing of primers for PCR, using its binding property with any natural
base.
 Adenosine deaminases acting on tRNA (ADAT) catalyse the conversion of Adenine
to Inosine. ADAR (on RNA) is not yet reported.

7. Inosine

8. Dihydrouridine
 Dihydrouridine or DHU (C₉H₁₄N₂O₆, Molar mass 246.217 g/mol) is a pyrimidine
nucleoside which is a product of addition of 2 hydrogen atoms to a Uridine.
 It is a highly conserved modified base found in tRNA and rRNA from all the domains of
life as a nucleoside.
 Physiological role of DHU remains unknown. Dihydrouridine synthase (DUS) catalyzes
the formation of tRNA through reduction of uracil base with Flavin mononucleotide
(FMN) as a cofactor.
Dihydrouridine

9. Formation of Dihydrouridine from Uridine by DUS

10. Pseudouridine
• Pseudouridine (C₉H₁₂N₂O₆, molecular mass 244.20 g/mol) is an isomer of nucleoside
Uridine in which uracil is attached via carbon-carbon bond.
• This bond replaces carbon-nitrogen glycosidic bond.
• It is denoted by the symbolΨ.Ψ is found in all species and in many classes of RNA.
• It is formed by enzymes called Ψ synthases which isomerize Uridine residues.This process
is called pseudouridylation.
• Recent studies shows that it reduces radiation induced chromosome aberrations in
human lymphocytes.

11. Pseudouridine

12. Non-Standard Base Pairing
• During the elongation process, after the action of aminoacyl tRNA synthetases on tRNA the amino acid is bound
to it.
• After the binding is complete the tRNA has to bind to specific codon site.
• During the binding of tRNA to the mRNA, anticodon-codon interactions occur.
• At the 1st position of anticodon loop or 3rd position of codon loop, wobble base occurs.
• The anticodon loop contain ‘I’ base which is equivalent to non Watson-Crick bases or non-standard bases like
Uridine, Inosine and Dihydrouridine.
• If perfect Watson-Crick base pairing was present between codon and anticodons, there would be 61 tRNA
molecules, one for each amino acid.
• But there are fewer tRNAs because one tRNA can bind to more than one amino acid.
• The broader recognition can occur because of wobble position.
• The first and second bases of codon always formWatson-Crick base pairs.
• This results in the carrying of variety of amino acids by single tRNA.

15. • E.g. Phenyl alanine has two codon sequences UUU and UUC (5`->3`). Both are
recognized by tRNA having GAA (5`->3`) anticodon sequence.
• Any two codons with N-N-Pyrimidine sequence can be decoded by single tRNA
having either G in the wobble position. (G can bind to any pyrimidine C or U).
• Inosine with a highest non-standard binding capacity (with A,C or U).
• tRNAs with Inosine are heavily employed in translation of codons that specify a
single amino acid.
• E.g. four out of six codons (CUA, CUC, CUU and UUA) are all recognized by anti
codon 3`-GAI-5`. Both G-U and Inosine related bindings can be observed.

16. Significance of codon-anticodon interactions
andWobble Hypothesis.
• These interactions bring out the decoding the language of DNA to synthesize
proteins, which are the building blocks of metabolism.
• The generation of variety of enzymes helps in several biochemical reactions and
metabolic pathways.
• The degeneracy of the of the genetic code can be explained by wobble hypothesis.

17. References
Books
1. Lodish.et.al, Molecular Cell Biology, 7th Edition, 2013, Page No. 133 and 134.
2. Weaver R.F, Molecular Biology, 2nd Edition, 2002, Page No. 50-52.
Articles
1. Yu F.et.al, Molecular basis of Dihydrouridine formation on tRNA, Prac Natl Acad Sci
U.S.A. Dec 2011; 6;108(49):19593-8.
2. Crick F.H.C, Codon-Anticodon Pairing:TheWobble Hypothesis, J. Mol. Biol. (1996) 19,
548-555.
3. Monobe.et.al, β-Pseudouridine a beer component, reduces radiation-induced
chromosome aberrations in human lymphocytes. Mutation research 538 (2003) 93-99.