Lecture 14: The Genetic Code (Do further reading)

Question 1

What was all the rage in the 1940s?

Answer 1

Code breaking

Question 2

What is the genetic code?

Answer 2

The genetic code refers to the instructions contained in a gene that tell a cell how to make a specific protein.

Question 3

How did the British crack the enigma code?

Answer 3

Using a secret radio intercept stadium at Bletchely Park

Question 4

4 nucleotides….

Answer 4

20 amino acids

Question 5

What nomenclature did Bletchely Park use for describing code?

Answer 5

overlapping, degenerate

Question 6

Who founded the exclusive ‘RNA tie club’?

Answer 6

George Gamow

Question 7

When did George Gamow find the ‘RNA tie club’?

Answer 7

1954

Question 8

What did George Gamow propose about the genetic code?

Answer 8

That if a triplet encodes an amino acid, proteins can contain 64 amino acids. As there are only 20 amino acids, the genetic code must be generate, with multiple triplets encoding for the same amino acid. This led to the diamond hypothesis.

Question 9

Describe the diamond hypothesis

Answer 9

If you take your double stranded helix and count the number of shapes that can be formed by the position of the nucleotides, there are 20 possible uniquely shaped pockets and 20 amino acids. He therefore came to the conclusion that the genetic code was overlapping, degenerative, and a triplet code.

Question 10

Was Gamow’s diamond hypothesis correct or incorrect?

Answer 10

Incorrect. This is because he used no experimental evidence, just maths.

Question 11

Who challenged Gamow and discovered that the genetic code was non-overlapping?

Answer 11

Sidney Brenner

Question 12

When did Sidney Brenner discover that the genetic code was non-overlapping?

Answer 12

In 1957

Question 13

What made Sidney Brenner realise that the genetic code was non-overlapping?

Answer 13

If the code overlapped, the amino acid encoded by UUA would always be followed by an amino acid encoded by UAA, UAG, UAC or UAU. Thus certain amino acid combinations should be over-represented – and some combinations would be impossible.

1957: Sidney Brenner compared the known amino acid sequences of proteins. Each amino acid could be found next to each of the other 19 amino acids. Conclusion: the code cannot be overlapping.

Question 14

Who coined the word cistron?

Answer 14

Seymour Benzer

Question 15

What is the meaning of Cistron?

Answer 15

Gene

Answer 16

• worked on a T4 bacteriophage
• this phgae infected e coli
• e coli then supported the generation of the next generation of phages by bursting and infecting other e colis around it
• wild type phages are cloudy and can infect E coli K and B strains
• the rII mutatnts (which he chose to focus on) are clear and lyse a lot more rapidly
• rII T4 cannot complete their life cycle in E. coli K strains but can in E. coli B strains.
• Realised that he could find out how far mutants are apart by recombining them in E coli
• He found that the recombination of rII mutant 1 and rII mutatnt 2 produced progeny that included: parental rII, double mutant, wild-type, parental rII
• He realised that the further apart the mutations were, the more likely you would be to get recombination between them
• He could work out how much recombination there was by growing all of the progeny on E coli B
• Then grow the progeny on E coli K and only the wild type will grow.
• Then this equation (RF = 2c/(a+b+c+d)
  ) will give you the recombination frequency.
• Frequency will be high if the mutations were far apart and will be low if mutants are close together.
• Realised he could map the position of mutants by measuring recombination frequency..

Answer 17

• Mapped over 2000 different mutations in the rII gene and realised that there were 2 genes/citrons
• Gene A is a gene that if you have mutations in it, you generate rapid lysis mutants
• If you get mutations in Gene B, you get rapid lysis
• There was a gap in gene B where he couldn’t find any mutations, so he came to the conclusion that this was a non essenital part of gene B, and so if this part was mutated, there would be no effect.

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Answer 18

• Wanted to go further than Seymour Benzer and see if they could actually work something out about the genetic code.
• If you have an area in gene B and you can’t detect any mutations, guessed that you could detect r+! or r-1 mutations. (addition of a base or deletion of a base).
• Argued that if you had a non-overlapping triplet code, that would give a reading frame. If you added a +1 or a -1, the reading frame would be disrupted.
• Proposed that acridine dyes such as PROFLAVIN added took away a base, causing a frameshift and everything downstream to be nonsense.
• They introduced a mutation by adding a base
• They retreated this mutation with proflavin
• This resulted in revertatnts to r+
• Crick and co then separated the mutations by recombination
• Thwy crossed a double mutant revertant 2 FCO with w2 and infected it into E coli B.
• They got the rare recombination and 4 products, one of which was the reverting mutation.
• The collected revertants and isolated as many as possible.
• +3 or -3 restored the reading frame: an experimentally determined triplet genetic code.

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Question 19

Who was despised by the RNA tie club?

Answer 19

Marshall Nirenberg

Question 20

What did Marshall Nirenberg do in 1961?

Answer 20

• He took an enzyme called polynucleotide phosphorylase and he gave it ribo UDP and let the enzyme make a poy(U)RNA.
• He then took cell extracts of E coli and added the poly(U) RNA to each one.
• He had 20 tubes including: the E coli extract, poly (U) RNA, 19 amino acids, and one radiolabelled amino acid.
• He incubated and acid-precipitated to look for incorporation of the radioactive amino acid into protein.
• In the tube with the radioactive amino acid, he got a radioactive protein.
• Solved the genetic code experimentally and revealed that UUU is a CODON that encodes phenylalanine (F). (first codon to be discovered).

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Question 21

What codons did Nirenberg publish?

Answer 21

• UUU encodes phenylalanine (F)
• CCC encodes proline (P)
• AAA encodes lysine (K)

Question 22

Who provided experimental eveidence that the code was degenerate?

Answer 22

Nirenberg

Question 23

Who provided experimental evidence for the central dogma?

Answer 23

Har Gobind Khorana

Question 24

How did Har Gobind Khorana provide evvidence for the central dogma?

Answer 24

He chemically synthesised DNA of KNOWN sequence.

Copied this into RNA using RNA polymerase .

And examined incorporation of radiolabelled amino acids into new protein.

Showed the flow of information from DNA to RNA to protein

e.g. ACACACACACACACAC gave polypeptides with equal amounts of threonine and histidine .

Question 25

What are the stop codons?

Answer 25

UAA

UAG

UGA

Question 26

Who proposed the idea that there was a tRNA?

Answer 26

Francis Crick. He proposed that there was an adaptor molecule as an intermediate between a messenger RNA and the protein and sets of these adaptors would therefore translate the information into protein.

Since base-pairing would allow specific adaptors to find the message, he proposed that the adaptor would be a nucleic acid

Question 27

In which direction do tRNAs read bases?

Answer 27

3’ to 5’

Question 28

Is thymine found in tRNA (as well as in DNA)?

Answer 28

Yes

Question 29

What shape do tRNAs fold into?

Answer 29

tRNAs fold into L shaped structures that expose the anticodon loop

Question 30

Who wanted to know why the genetic code was dgenerate?

Answer 30

Francis Crick, as a result he proposed his ‘wobble hypothesis’.

Question 31

Describe Francis Crick’s wobble hypothesis

Answer 31

① The first two bases of the codon pair with the last two bases of the anticodon, and confer most of the coding specificity

② The first base of the anticodon can ‘wobble’ and determines the number of codons that can be recognised by the tRNA.

③ When an amino acid is specified by several different codons, codons that differ in either of the first two bases require different tRNAs

④ A MINIMUM of 32 tRNAs are required to translate all 61 codons (31 encode amino acids and there is a special initiator tRNA).

Question 32

Whilst there are some minor differences to the genetic code, for example in mitochondria, in general, do any organisms have a completely different genetic code?

Answer 32

No

Question 33

What enzyme is there for every tRNA?

Answer 33

tRNA synthetases

Question 34

What does the synthase carry?

Answer 34

A specific amino acid. 2 sites, one which will carry a specific tRNA and one that will carry a specific amino acid.

Question 35

What is an uncharged tRNA written like?

Answer 35

Uncharged tRNA is written: tRNAaa e.g. tRNALeu

Question 36

What is a charged tRNA written like?

Answer 36

Charged tRNA is written: aa-tRNAaa e.g. Leu-tRNALeu

Question 37

Is it true that Aminoacyl tRNA synthetases can also edit, replacing an incorrectly chosen amino acid with the correct choice?

Answer 37

Yes

Question 38

What is required to charge a tRNA?

Answer 38

Energy

Answer 39

• ATP is hyrdolysed to AMP to release pyrophosphate.
• This results in the energy required for a bond to be formed between the amino acid and the acceptor site of the tRNA.

Question 40

Summary

Answer 40

1. Codons are triplets of bases: they do not overlap
2. The genetic code is degenerate. This means that each amino acid is specified by between one and six codons (There are only 20 amino acids and 64 possible codon triplets).
3. The code includes punctuation in the form of three “stop” codons that do not code for an amino acid:
   UAA, UAG, and UGA
4. The initiation codon is (almost always) AUG (methionine).
5. The mRNA strand is read from the 5’ to the 3’ end.

6.
If there are mutations or errors in the DNA, the message may be changed and incorrect protein formation results.

7.
Highly specific tRNA: amino acid combinations translate the code, matching CODONs with specific amino acids.