25 - The Genetic Code

Question 1

What were the first three kinds of genetic code proposed by Barnett, Brenner and Francis Crick with T4 phages? Which one fit all all experimental observations and was determined correct?

Answer 1

• Overlapping code (regions of codons overlapping)
• Punctuated code (bases between codons)
• Unpunctuated code (correct)

Question 2

How was it determined that RNA can serve as messenger? What did this prove?

Answer 2

• A cell free protein synthesizing system (translation extract) was prepared from E. coli extracts and incubated for mRNA degradation and DNase incubation to insure no mRNA made
• Polynucleotide phosphorylase was used to synthesize RNA templates of defined sequence
• The RNA template was given to translation extract and the resultant polypeptide was repeats of uracils which coded for phenylalanine repeats (polyphenylalanine)
• This also proved that UUU coded for phenylalanine (the first codon to be deciphered)

This proved that RNA acted as the messenger (mRNA) to bring genetic info from DNA to protein

Question 3

Random polyribonucleotides can be synthesized from ribonucleoside diphosphates (rNDPs) by what enzyme? How do we know?

Answer 3

Polynucleotide phosphorylase

• This is a reversible reaction that works to degrade cellular RNA in biological conditions.
• The enzyme does not require a template (template independent)
• If a single rNDP is used as substrate the product is a homopolymer (eg. UUUUUU)
• If more than one rNDP is used as substrate, than the product is a random copolymer (UAUAAUUUUA etc.)
• Base composition purely determined by ratio of rNDPs

Question 4

How were most codon compositions determined for amino acids?

Answer 4

By using copolymers as templates (eg. adding rUDP and rGDP in a specific ratio) and using polynucleotide phosphorylase to synthesize random copolymers. Than the relative proportion of triplets were associated with the observed relative incorporation of amino acids

Question 5

How were synthetic polyribonucleotides with repeating sequences used to decipher codons and confirm the triplet code?

Answer 5

The polymer can specify three different polypeptides, depending on which reading frame is used. These would yield different polypeptides for one strand of polyribonucleotides (eg. polylysine, polyglutamate etc.)

Question 6

How were specific aminoacyl-tRNA molecules found for specific codons/amino acids?

Answer 6

Trinucleotide sequences (single codons) were produced and treated with a radioactive aminoacyl-tRNA and ribosome mixture. Then passed through a nitrocellulose filter.

If the tRNA was specific for the codon, then they would bind to a ribosome and would be retained on the nitrocellulose. Free aminoacyl-tRNAs would not be retained. A positive match would be observed as radioactivity retained on the nitrocellulose.

Question 7

Chemically synthesized polyribonucleotides of defined sequence direct the synthesis of polypeptides of defined sequence. How did H. Gobind Khorana use this method to confirm the codon assignments established previously?

Answer 7

Observed that the same alternating sequence of two amino acids (eg. Cys and Val) results regardless of where translation begins

Question 8

How were termination codons discovered?

Answer 8

Synthetic polyribonucleotides with repeating sequences were translated and resulted in either homopolypeptides (eg. poly(Val)) or no incorporation of any amino acids. The sequences with no incorporation of any amino acids were determined to be nonsense codons and signals for the stop of translation.

Question 9

How many possible triplet combinations of the four nucleotides are there and how many encode for individual amino acids?

Answer 9

• 64 possible triplet combinations
• 61 encode for amino acids
• 3 are STOP codons

Question 10

By saying the genetic code is degenerate, what does that mean?

Answer 10

Most amino acids are specified by more than one codon

Question 11

Are codons written in DNA or mRNA?

Answer 11

mRNA

Question 12

What is the start codon?

Answer 12

AUG - Met - Methionine

Sometimes UUG (leucine) or AUU (isoleucine), which when used as start codons will code for Met

Question 13

When AUG or other codons are used as start codons, what type of amino acid do they specify for in prokaryotes and eukaryotes?

Answer 13

Prokaryotes: N-formylmethionine (N-fMet)

Eukaryotes: unformylated Met

Question 14

What does it mean when we say the genetic code is unambiguous?

Answer 14

One codon for one amino acid

Question 15

The base at the ___ position in the anticodon pairs with the base at the __ position in the codon

Answer 15

The base at the 5’ position in the anticodon pairs with the base at the 3’ position in the codon

Question 16

List what these bases in anticodons bind with in codons:

C
A
G
U
I

Answer 16

C: G
A: U
G: C/U
U: A/G
I: A/U/C

Question 17

What are two biological purposes for degeneracy in the genetic code?

Answer 17

• To minimize the deleterious effect of mutations (degeneracy provides mostly silent mutations as it results in the replacement of an amino acid rather than a nonsense STOP codon)
• Similar protein sequences can be synthesized in spite of wide differences in the base composition of the genome encoding them

Question 18

What is evidence that the genetic code in universal?

Answer 18

Most substitutions across organisms are explained by a single base change, codon changes across organisms are such that the same codon is seen to code for the same amino acid (eg. GAA for Glutamate)

Question 19

What is one exception to the universality of the genetic code? What is this called?

Answer 19

IN animal and fungal mitochondria, UGA specifies Trp rather than STOP

Called a non-standard codon assignment