Lecture 14: The Genetic Code [G](Do further reading)

Question 1

What was all the rage in the 1940s?

Answer 1

Code breaking. The efforts at Bletchley Park (e.g., cracking the German Enigma code) inspired geneticists.

Question 2

DNA -> RNA -> Proteins

Answer 2

DNA -> RNA -> Proteins

Question 3

What is the genetic code?

Answer 3

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

Question 4

How did the British crack the enigma code?

Answer 4

Using a secret radio intercept stadium at Bletchely Park

Question 5

4 nucleotides….

Answer 5

20 amino acids

Question 6

What nomenclature did Bletchely Park use for describing code?

Answer 6

overlapping, degenerate

Question 7

Who founded the exclusive ‘RNA tie club’?

Answer 7

George Gamow

Question 8

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

Answer 8

1954

Question 9

What did George Gamow propose about the genetic code?

Answer 9

Proteins are made from 20 amino acids, but a triplet code (3 nucleotides) gives 64 possible combinations (4 × 4 × 4 = 64). Since there are more combinations than amino acids, this means that some triplets must code for the same amino acid. This is what we call a degenerate code.

Initially, scientists thought the genetic code might be overlapping (one nucleotide could be part of multiple triplets), but this turned out to be wrong. Each triplet is read separately, one after the other.

Question 10

Describe the diamond hypothesis

Answer 10

• 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 11

Was Gamow’s diamond hypothesis correct or incorrect?

Answer 11

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

Question 12

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

Answer 12

Sidney Brenner

Question 13

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

Answer 13

In 1957

Question 14

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

Answer 14

• 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 15

Who coined the word cistron?

Answer 15

Seymour Benzer

Question 16

What is the meaning of Cistron?

Answer 16

Gene

Question 17

What analytical procedure did Seymour Benzer use to understand the structure of the rII locus of the T4 bacteriophage?

Answer 17

• Worked on a T4 bacteriophage and induced mutations in it in the rII locus of the T4 bacteriophage.
• He infected E coli with the mutated T4 bacteriophage.
• 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..
• This work provided one of the earliest and most detailed insights into the fine structure of genes and laid the foundation for understanding the molecular nature of mutations and genetic recombination. It also showed that genes could be studied at a resolution approaching the level of individual

Question 18

Seymour Benzer

Answer 18

• 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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Question 19

Crick, Barnet, Brenner and Watts-Tobin

Answer 19

• 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+1 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 revertants to r+
• Crick and co then separated the mutations by recombination
• They 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.
• Demonstrated that the genetic code is read in triplets.

Question 20

Who demonstrated that the genetic code is read in triplets?

Answer 20

Crick, Barnet, Brenner and Watts-Tobin

Question 21

Who was despised by the RNA tie club?

Answer 21

Marshall Nirenberg

Question 22

What did Marshall Nirenberg do in 1961?

Answer 22

• An enzyme called polynucleotide phosphorylase was used to synthesize RNA. The enzyme was supplied with ribonucleotide diphosphates (ribo UDP), which resulted in the production of a polymer consisting entirely of uracil bases, known as poly(U) RNA.
• He then took cell extracts of E coli and added the poly(U) RNA to each one.
• E. coli cell extracts (containing the necessary machinery for protein synthesis) were prepared.
• The poly(U) RNA was added to each extract to serve as the template for protein synthesis.
• The experiment used 20 test tubes, each containing: The E. coli extract, Poly(U) RNA, A mix of 19 unlabeled amino acids, One radiolabeled amino acid (different in each tube).
• The tubes were incubated to allow protein synthesis to occur.
• Afterward, the mixtures were treated with acid to precipitate any proteins formed, separating them from unincorporated amino acids.
• The precipitates were analyzed to detect the incorporation of the radioactive amino acid into newly synthesized protein.
• In the test tube containing radiolabeled phenylalanine, a radioactive protein was detected. This demonstrated that the codon UUU specifically encoded the amino acid phenylalanine (F).
• This experiment was the first to decode a codon and provided direct evidence of the relationship between specific RNA sequences and the amino acids they encode. It marked the beginning of cracking the genetic code.

Question 23

What codons did Nirenberg publish?

Answer 23

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

Question 24

Who provided experimental eveidence that the code was degenerate? (multiple codons can code for the same amino acid)

Answer 24

Nirenberg

Question 25

Who provided experimental evidence for the central dogma?

Answer 25

Har Gobind Khorana

Question 26

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

Answer 26

• 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 27

What were the achievements of Har Gobind Khorana ?

Answer 27

• Completed the codon table.
• Confirmed degeneracy (e.g., multiple codons encoding the same amino acid).
• Discovered stop codons (UAA, UAG, UGA).

Question 28

What are the stop codons?

Answer 28

UAA

UAG

UGA

Question 29

Who proposed the idea that there was a tRNA?

EXPLAIN

Answer 29

• 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 30

In which direction do tRNAs read bases?

Answer 30

3’ to 5’

Question 31

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

Answer 31

Yes

Question 32

What shape do tRNAs fold into?

Answer 32

tRNAs fold into L shaped structures that expose the anticodon loop

Question 33

Who wanted to know why the genetic code was dgenerate?

Answer 33

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

Question 34

Describe Francis Crick’s wobble hypothesis

Answer 34

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). This reduces the number of tRNAs needed

Question 35

Is it true that Crick’s wobble hypothesis ‘allows tRNA flexibility, reducing the total number of tRNAs needed’ ?

Answer 35

Yes

Question 36

What are Aminoacyl-tRNA synthetases?

Answer 36

Enzymes that ensure the correct matching of amino acids with their respective tRNAs, which is essential for accurate protein synthesis.

Question 37

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 37

No

Question 38

What enzyme is there for every tRNA?

Answer 38

tRNA synthetases

Question 39

What does the synthase carry?

Answer 39

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

Question 40

What is an uncharged tRNA written like?

Answer 40

Uncharged tRNA is written: tRNAaa e.g. tRNALeu

Question 41

What is a charged tRNA written like?

Answer 41

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

Question 42

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

Answer 42

Yes

Question 43

What is required to charge a tRNA?

Answer 43

Energy

Answer 44

• 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 45

Summary

Answer 45

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.