Lecture 14: The Genetic Code

Question 1

Who shared the Nobel prize for approaches in understanding genetic code?

Answer 1

Marshall Nuremberg and Har Gobind Khorana

Question 2

Describe the work of George Gamow and the RNA tie club

Answer 2

• suggested triplet code must be triplet and degenerate
• formed RNA tie club to crack code mathematically
• proposed diamond hypothesis where each AA has a distinct diamond pocket
  -> completely wrong but introduced concept of overlapping, degenerate triplet code

Question 3

What did Sidney Brenner discover?

Answer 3

• if code overlapped, certain amino acids would be over-represented and some would be impossible
• compared known protein sequences and determined that all amino acids can appear next to any other - non-overlapping triplet code

Question 4

Work of Seymour Benzer and what it showed

Answer 4

• rII mapping in T4 bacteriophage
  -> r+ (slow lysis) - turbid plaques on E.coli K and B
  -> rII (rapid lysis) - clear plaques on E.coli B
• RF mapping showed many rII mutants
• showed rII region had two genes
• identified essential and nonessential region

Question 5

RF calculation

Answer 5

2c / a + b + c + d

Question 6

Describe Crick, Brenner, Barnett & Watts-Tobin frameshift experiment

Answer 6

• acridine dyes are mutagens that cause single nucleotide additions or deletions
  -> cause +1 or -1 mutations
• logic was that a single base addition would cause a frameshift leading to rII phenotype, and that secondary mutation would restore frame
• found that +1,-1 is always frameshit (rII phenotype)
• +3/-3 restored the reading frame (r+ phenotype)
  -> therefore code is read in triplets

Question 7

How did Nirenberg and Matthaei contribute to cracking code (Nobel prize)

Answer 7

• used polynucleotide phosphorylase and UDP to make synthetic poly U RNA
• added polyU to E.coli and 20 amino acids (one radioactive)
• determined code for F (UUU) and P and K
• able to determine that condones with A & C code for N, Q, H, T, P, K

-> code is degenerate!

Question 8

Describe Khoranas definitive synthetic gene work (Nobel prize)

Answer 8

• chemically synthesised DNA with known repeating sequences
• transcribed RNA, translated it and analysed protein incorporation
• got CAC as His and ACA as Thr
  -> he demonstrated flow of info from DNA to RNA to protein (central dogma)

Question 9

Describe the universal genetic code

Answer 9

• 64 codons
• 61 sense codons
• 3 stop codons (UAA, UAG, UGA)
• initiator codon is AUG

Question 10

Describe Cricks adaptor hypothesis

Answer 10

• proposed existence of adaptor molecules that:
  -> base pair with mRNA
  -> carry correct amino acid
• later identified as tRNA

Question 11

Describe the structure of tRNA

Answer 11

• 73-93 nucleotides
• L shaped
• D loop
• anticodon loop that matches codon
• TpsiC loop - presence of T
  -> psi is a modified base
• 3’ CCA tail which is site of amino acid attachment

Question 12

Why are amino acids degenerate?

Answer 12

• largely due to the 3rd base of the codon being less important

Question 13

What is Cricks wobble hypothesis and what does it show?

Answer 13

• explain why the genetic code is degenerate—why many amino acids are encoded by more than one codon.
• only the first two bases of the mRNA codon require strict Watson–Crick base pairing with the last two bases of the tRNA anticodon.
• the third base of the codon (and the first base of the anticodon) can “wobble,” allowing non-standard base pairing.

-> flexibility allows one tRNA to recognise multiple codons.

Question 14

What is the role of aminoacyl tRNA synthetases?

Answer 14

• one synthetase per amino acid
• recognises the tRNA anticodon and the correct amino acid
• incorrect amino acids edited out via editing site

Question 15

Notation for charged and uncharged tRNA

Answer 15

Uncharged: tRNA**
Charged: aa-tRNA**

Question 16

Describe how energy is used in aminoacylation

Answer 16

• amino acid + ATP —> aminoacyl-AMP + PPi
• amino acid transferred to 3’ CCA of tRNA
• aminoacyl-tRNA is an activated amino acid