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Genes in Action: Standard genetic code and how it was deciphered. Near universality of the code.

Terms in this set (32)

During world war 2, Bletchley park established the nomenclature for describing codes: overlapping, degenerate etc. In the 1950s, the coding craze continued, at the time it was known that...
> a string of nucleotides, in a 4 letter alphabet, encoded a in a double helix...
> was transcribed into a working copy (mRNA postulated but not yet discovered)
> which was translated into an amino acid sequence of a 20 letter alphabet.
- people wanted to know how this information was encoded and decoded.
George Gamow formed the exclusive 'RNA Tie Club' in 1954. It's believed he postulated that a triplet code must encode each amino acid, what led him to this?
- if 1 nucleotide encodes 1 amino acid, proteins could only encode 4 amino acids.
- if a nucleotide pair encodes 1 aa, proteins can only encode 16 (4×4) aa.
- if a triplet encodes 1 aa, proteins could encode 64 (4×4×4).
∴ minimal coding unit must be a triplet, and the code must be degenerate
- led to the diamond hypothesis
What was George Gamow's rationale behind the 1954 diamond hypothesis?
- each amino acid would fit directly into distinct diamond shaped pockets formed within the grooves of DNA where the 4 sides of each pocket would be defined by 4 bases.
- there's 20 unique pockets, and 20 amino acids.
- Gamow concluded that code was a triplet code, which was degenerate and overlapped.
Who tested the Diamond Hypothesis?
1957: Sydney Brenner
- if the code overlapped, certain amino acid combinations should be over-represented - and some combinations would be impossible.
- 1957: compared the known amino acid sequences of proteins. Each amino acid could be found next to each of the other 19 aa.
- concluded the code can't be overlapping.
1959 - 1960 an analytical procedure to map the genetic structure of a gene was developed by Seymour Benzer. With what molecule did he do this with?
- mapped the rII locus of the T4 bacteriophage.
- if T4 infects E.coli, a wild type,r⁺, (slow lysis) turbid plaque is produced, with no clear boundary. r⁺ can infect E.coli K and B strains.
- rII (rapid lysis) plaques are clear and have a distinct boundary. Can only complete life cycle in B strains.
How did Seymour Benzer map the genetic structure of a gene?
- realised you could recombine the mutations of rII, and then map them on the rapid lysis gene.
- if you co-infect E.coli B with 2 mutants, you produce a rare recombination.
- realised there'd be different growth on different strains.
Seymour Benzer showed that for any pair of mutants...
there'd be a characteristic recombinant frequency, depending on how far apart they were.
- Recombination Frequency = 2 × one that only grows on K / sum total
How many mutations did Benzer find?
~2000 in the rII gene, he mapped them in liner order (pairs of mutants with high RF are further apart, pairs with low RF are closer).
Seymour Benzer showed that the rII locus...
- has 2 coding regions he called 'cistrons' (A and B)
- each cistron acts as a gene that encodes a polypeptide
- part of cistron B can tolerate mutations (a non-essential part)
1961: Crick, Barnet, Brenner and Watts-Tobin: reasoned...
> the acridine dyes, e.g. PROFLAVIN, are mutagens, which either insert or delete 1 nucleotide on DNA.
> Crick, etal. reasoned that if a proflavin induced alteration (e.g. an addition) occurred in the non-essential region of the T4 r⁺ B locus, there'd be a FRAMESHIFT, all nucleotides downstream would be shifted right.
> this would produce a clear plaque (rII rapid lysis phenotype).
Describe Crick, et al. 1961 experiment.
1. wild type r⁺ gene was treated with Proflavin.
2. mutates the gene (Francis Crick Zero).
3. FC0 is remutated with Proflavin, revertants to wild type r⁺ would occur because a deletion in the non-essential region restores the downstream reading frame.
- Crick et al. could then separate mutations.
In the same way that Benzer combined mutations, Crick, et al. separated their mutations by recombination. Describe how.
1. take a double mutant revertant FC0 and a wild type, and make separate T4 phage containing the different alleles.
2. let the T4 phage co-infect E.coli B.
3. occasionally produce a rare recombination, if you resolve this recombination one would be:
> parental FC0 revertant
> parental wild type
> revertant 2 ← isolation of the reverting mutation
> FC0
4. Crick, et al. used these recombinations and then recombined them. They found that +3 or -3 restored the reading frame: an experimentally determined triplet code.
Marshall Nirenberg was ignored by George Gamow, but won the 1961 Nobel Prize. Nirenberg took polynucleotide phosphorylase, purified it and added UDP, the enzyme...
polymerased it into poly(U) RNA.
- poly(U) RNA was divided into 20 test tubes containing E.coli.
- incubated, and acid precipitated to look for incorporation of radioactive amino acid into protein.
- revealed that UUU is a codon that enodes phenylalanine.
- later developed the Codon Chart.
Marhsall Nirenberg attempted to make other codon designations. How did he do this?
- supplied polynucleotide phosphorylase with ADP and CDP in a 5:1 ratio to make mutliple RNAs composed of A and C residues.
- he calculated the frequency of the triplets in the RNA that was made, he found that there was more amino acids incorporated into the proteins than expected.
- ∴ these amino acids could be encoded by mutliple codons ∴ the code is degenerate.
- with this information he could fill out more of the Codon Chart.
Har Gobind Khorana chemically synthesised DNA of known sequence, adding one nucleotide at a time to two complementary strands and then hybridised them together. He copied this into...
RNA using RNA polymerase. He then examined the incorporation of radio-labelled amino acids into the protein.
> one combination was poly AC, which gave polypeptides with equal amounts of histidine and threonine.
- since His was already found to be encoded by a codon with 2Cs and an A, then:
CAC → His
ACA → Thr
> using this approach Khorona filled out the Codon Chart rapidly.
What did Har Gobind Khorona demonstrate?
Khorona demonstrated the central dogma of the flow of information from DNA to RNA to Protein.
What is was 'Crick's Adaptor Hypothesis'?
- proposed an adaptor molecule was an intermediate between a messenger RNA and the protein, and sets of these adaptors would ∴ translate the information into protein.
- at first this was shunned by many biochemists who believed that they would have already discovered this mechanism due to it's dependence on enzymes to join the amino acids.
Why did Crick, in his adaptor hypothesis, propose that the adaptor was likely to be a nucleic acid?
since base-pairing would allow specific adaptors to find the message
The adaptors that Crick theorised became known as transfer RNAs. Describe the structure of tRNA.
> ~73 - 93 nucleotides, 7-15 of which are modified.
> e.g. dihydrouridine.
> ~half the molecule is base-paired.
> 3-loops are conserved: the D loop, the T loop and the anticodon loop in a 'clover leaf' array.
> there's a variably sized 'extra loop' between the anticodon and T loops.
> the anticodon loop bears the anti-codon, which can base-pair with a codon.
> a specific amino acid (that matches the anti-codon sequence) is attached at the 3'-CCA motif.
> only known RNA to utilise thymidine.
tRNA fold, from their clover shaped secondary structure, into an...
L shaped structure that exposes the anticodon loop and presents the amino acid in the right orientation.
Met and Trp are encoded by...
single codons.
Ile is encoded by...
3 codons.
Adise from Met, Trp and Ile, the amino acids are encoded by...
2, 4 or 6 amino acids. The reason for this was proposed by Francis Crick in 1966, with the Wobble Hypothesis.
What does Crick's 1966 Wobble Hypothesis state?
1. codon's first 2 bases form always form strong Watson-Crick base pairs with the last two bases of the anticodon, and confer most of the coding specificity.
2. anticodon's first base 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 require different bases that differ in either of their first two bases.
4. minimum of 32 tRNAs are needed to translate all 61 codons (31 encode amino acids and theres a special initiator tRNA).
What are Crick's wobble rules?
No Wobble -
C: pairs only with G. 1 codon only.
A: pairs only with U. 1 codon only.
Wobble -
U: pairs with A or G. 2 codons.
G: pairs with C or U. 2 codons.
I: inosine pairs with A, U or C. 3 codons.
What direction is the mRNA strand read?
5' → 3'
To read each gene and select necessary information to form proteins, cells begin at a fixed and specific starting point on the mRNA strand. What is the initiation codon?
AUG
What are the 3 'STOP' codons?
UAA
UAG
UGA
Charging tRNA requires energy. Describe how tRNA is charged.
1. charging tRNA^aa requires the hydrolysis of ATP to release energy.
2. energy released is used to form a HIGH ENERGY BOND between tRNA^aa and it's amino acid.
How does the nomenclature for charged tRNA?
uncharged tRNA written: tRNA^aa
charged tRNA written: aa-tRNA^aa
aa = 3 letter code
How is tRNA charged?
> aminoacyl tRNA synthetases exist for every amino acid.
> they have 2 binding sites, one of which can bind to a specific anticodon on tRNA, the other binds to the corresponding amino acid.
> aminoacyl tRNA synthetases can also edit, replacing an incorrectly chosen amino acid with the correct choice.
There are 4 minor differences from the standard genetic code, explain why.
> some are caused by slightly altered 'wobble rules', others may be caused by codons coding for different amino acids in different organisms, for example.
> No examples of a completely different code have been found, implying that all life is connected by the genetic code.
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