The genetic code

Question 1

Knowledge genetic code underlies molecular biology

Answer 1

Used day to day to sequence proteins and determine amino acids protein functions
Protein functions and structures can with protein sequence data
Key residues can be mutated to address protein function
Genetic code understood before techniques developed for isolation genes and sequencing DNA

Question 2

Coding sequences have 3 different reading frames

Answer 2

Specific codons define beginning and end of ORF
- Initiation codon ~ AUG (encodes for methionine)
- termination codons ~ UAA, UAG, UGA
Codons said be consecutive, non punctuated, non overlapping
Are three different possible reading frames each RNA sequence
Initiation and termination codon determine the reading frame
Start translation doesn’t always start transcription, transcription can start halfway down RNA sequence

Question 3

Cracking genetic code - part 1: in vitro translation

Answer 3

Ribosome can be isolated from cell extract and programmed to make proteins using artificial RNAs
First experiment:
- poly(U) RNA shown specifically direct incorporation of phenylalanine into polypeptide
- Codon UUU deduced encode phenylalanine
- Already deduced codons read in triplets
- Used radiolabelled RNA to deduce sequence
Later experiments:
- Tested other homopolymers and heteropolymer RNAs
- Quite difficult tell which sequence caused what in heteropolymers

Question 4

Cracking genetic code - part 2: ribosome binding

Answer 4

This expreiment established genetic code
Bind “charged”
Need to go back over this

Question 5

The genetic code

Answer 5

All codon code for something
61 codons code specific amino acid
- Some unique (start)
- Some degenerate (multiple codon = same amino acid)
Synonymous codons usually differ at 3rd nucleotide
First 2 nucleotides v important for determining amino acid
Related codons encode similar amino acids

Question 6

Non-Watson-Crick base-pairing at 3rd base

Answer 6

Not all organism by 61 different tRNAs
tRNAs can bp with more than 1 codon due to “wobble” - tRNAs can recognise more than 1 trinucleotide sequence
Many tRNAs have modified nucleotides at 1st position of anticodon
G in position1 can recognise codon ending with C or U
Inosine bp with A, C or U
tRNA ^Leu(down again)(IAG) can recognise CUA, CUC, CUU and UUA

Question 7

tRNAs have “cloverlead” secondary structure

Answer 7

5’ and 3’ ends drawn together
Amino acid attached to 3’ hydroxyl group of 3’ terminal A nucleotide
Variable arm different in each tRNA
Specific nucleotides within tRNAs post-transcriptionally modified
Modification of 1 position of anticodon allows “wobble”

Question 8

tRNAs folded into L-shape

Answer 8

Tertiary structure tRNA
Coaxial stacking of helicase and bpairing between end of D (2 loop) and TψC (4 loop) loops produce flat L-shapped molecule
Anticodon loop and aminoacul group positioned at opposite ends of molecules

Question 9

Chargin tRNAs

Answer 9

tRNAs “charged” with appropriate amino acid by aminoacul-tRNA synthetases
Amino acid added to 3’ position on tRNA
Single aminoacyl tRNA synthetase charges all isoacceptor tRNAs
Reaction requires ATP
Amino acid linked to tRNA by ester linkage between carboxylic acid group of amino acid and 3’ hydroxyl group of terminal nucleotide