1. Translating the Genetic Code
Gene expression part 3

2. An overview of gene expression
Figure 13.2

3. The Idea of A Code 20 amino acids 4 nucleotides
How do nucleic acids composed of 4 nucleotides specify the synthesis of proteins composed of 20 different amino acids?
Figure: 12-01
Caption:
Flow of genetic information encoded in DNA to messenger RNA to protein.

4. The Coding Problem 1-1 correspondence – NO – 4 possibilities
3-1 correspondence – YES – 64 possibilities

5. Determination of Triplet Nature of Code
Aacridines & flavinoids cause single nucleotide deletions and insertions respectively
Insertion of 1 base shifts frame & creates non-sense mutation
Deletion of base in mutant restores frame (suppresses 1st mutation)
Recombination between mutants showed that insertion of 1 or 2 bases or removal of 1 or 2 bases doesn't restore frame, but insertion of 3 or removal of 3 allows frame to continue
Figure: 12-02ab
Caption:
The effect of frameshift mutations on a DNA sequence repeating the triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then reestablished to the original sequence.

6. Crick’s Experiments to Determine Triplet Nature of Code

7. So.. The code is 3 letter words, but what about punctuation?
GROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c
There is a message, but one must start at the right place to read it
Code written in three letter words - codon
There are three reading frames, but only one gives an intelligible message – frame b
NOW THE CAT SAW THE DOG BUT DID NOT RUN
A start codon (NOW) and a stop codon (END) define the frame to use

8. Any frame is potentially the reading frame!
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c
OWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c
The actual reading frame is called the Open Reading Frame or ORF

9. Reading Frames & Mutation Types
Frame shift mutations
Original reading frame is frame a
Insertions or deletions shift the reading frame
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c
ROWNDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c ^

10. Reading Frames & Mutations
ROWANDNOWTHECATSAWTHEDOGBUTDIDNOTRUNENDSEW a b c
Once a ribosome begins translation in a particular frame (a) it does not shift frames
Therefore, if a mutation shifts the reading frame in the mRNA, the ribosome will read the wrong frame. ^
ROWANDNOWTHECATSAWTHEADOGBUTDIDNOTRUNENDSEW a b c
NOW THE CAT SAW THE ADO GBU TDI DNO TRU NEN DSE W..

11. Deciphering the Code
Each amino acid in a protein is specified by 3 nucleotides of codon
Each codon specifies only ONE amimo acid
There are 64 possible codons but only 20 amino acids
Degeneracy
An amino acid can be specified by multiple codons
A given codon still only specifies only one amino acid

12. Deciphering the Code: Three Approaches
Synthesis and translation of homopolymer RNA & ratio polymer RNA followed by amino acid analysis
Synthesis and translation of di, tri and tetra nucleotide repetitive RNA polymers followed by amino acid analysis
Triplet RNA-tRNA binding assay and amino acid analysis

13. Synthetic RNA Templates and In Vitro Translation
Figure: 12-03
Caption:
The reaction catalyzed by the enzyme polynucleotide phosphorylase. Note that the equilibrium of the reaction favors the degradation of RNA but can be “forced” in the direction favoring synthesis.
Synthesis of homopolymeric RNAs
UUUUUUUUUU, AAAAAAAAAA, CCCCCCCCC, & GGGGGGGGG
When translated produced polypeptides
poly phenylalanine (UUU), polyproline (CCC), polylysine (AAA), and polyglycine (GGG)
Therefore 4 codons were determined
UUU = phe
CCC = pro
AAA = lys
GGG = gly

14. Decoding the Genetic Code
The enzyme polynucleotide phosphorylase
polymerizes ribonucleoside diphosphates (NDPs) into RNA
It does not use a template, the order of nucleotides is random
The nucleotide sequence is controlled by the relative abundance of NDPs
For example, if 70% GDP and 30% UDP are mixed together, then …
Radiolabeled Amino Acid Added
(%) Amino Acid Incorporated
Glycine (GGG + GGU, UGG, GUG)
49 ( )
Valine (GGU, UGG, GUG, GUU, UUG, UGU)
21 (15 + 6)
Tryptophan (UGG, GUG, GGU) 15
Cysteine (UUG, GUU, UGU) 6
Leucine (UUG, GUU, UGU)
Phenylalanine
(UUU) 3
Possible Codons
Percentage in the Random Polymer
GGG
0.7 x 0.7 x 0.7 = 0.34 = 34%
GGU
0.7 x 0.7 x 0.3 = 0.15 = 15%
GUU
0.7 x 0.3 x 0.3 = 0.06 = 6%
UUU
0.3 x 0.3 x 0.3 = 0.03 = 3%
UGG
0.3 x 0.7 x 0.7 = 0.15 = 15%
UUG
0.3 x 0.3 x 0.7 = 0.06 = 6%
UGU
0.3 x 0.7 x 0.3 = 0.06 = 6%
GUG
0.7 x 0.3 x 0.7 = 0.15 = 15%

15. Polymers of Nucleotide Repeats

16. Trinucleotide-tRNA Binding Analysis
Figure: 12-05
Caption:
An example of the triplet binding assay. The UUU triplet acts as a codon, attracting the complementary tRNAphe anticodon AAA.

17. Figure: 12-07
Caption:
The coding dictionary. AUG encodes methionine, which initiates most polypeptide chains. All other amino acids except tryptophan, which is encoded only by UGG, are represented by two to six triplets. The triplets UAA, UAG, and UGA are termination signals and do not encode any amino acids.

18. The code is nearly universal
Special codons:
AUG (which specifies methionine) = start codon
AUG specifies internal methionines also
UAA, UAG and UGA = termination, or stop, codons
The code is degenerate
More than one codon can specify the same amino acid
For example: GGU, GGC, GGA and GGG all code for lysine
In most instances, the third base is the degenerate base
It is sometime referred to as the wobble base
The code is nearly universal
Only a few rare exceptions have been noted