Download Visualizing the triplet code

Visualizing the triplet code
Reading Frame:
1
2
3
THE FAT CAT ATE THE BIG RAT
HEF ATC ATA TET HEB IGR AT
EFA TCA TAT ETH EBI GRA T
add or delete one base ===> frameshift
THE FAN TCA TAT ETH EBI GRA T
THE FAC ATA TET HEB IGR AT
Visualizing the triplet code
Implications of the code from the rII experiment
If you add (or delete) 3 nucleotides you get back to the same reading frame
But…….the sequence will encode additional, fewer or different amino acids
But………what is the code?
How does each 3 base codon translate into the 20 amino acids?
Key innovations
1. In vitro translation
2. Synthetic nucleic acids
RNA
protein
cell extract
ribosomes
amino acids
tRNA
5’-AAAAAAAAAAAAAAAAAAAAAAAA-3’
5’-CCCCCCCCCCCCCCCCCCCCCCCC-3’
5’-GGGGGGGGGGGGGGGGGGGGGGGG-3’
5’-UUUUUUUUUUUUUUUUUUUUUUUU-3’
But………what is the code?
Nirenberg and Matthei
5’-UUUUUUUUUUUUUUUUUUUUUUUU-3’
RNA
(polyU)
protein
only 14C phe
resulted in
labeled protein
14C
radiolabeled
amino acids
Nobel Prize, 1968
UUU
AAA
CCC
GGG
–
–
–
-
phenylalanine
lysine
proline
glycine
But………what is the code?
Other key experiments/findings:
1. Synthetic RNA made with dinucleotides, trinucleotides etc.
- (e.g. 5’-UCUCUCUCUCUCUCU-3’ encodes poly Leu-Ser)
- H. Gobind Khorana – Nobel Prize, 1968
2. Matching trinucleotides with amino acids
3. Point mutations leading to single amino acid substitutions
- Charles Yanofsky, trp operon in E. coli
4. Point mutation leading to a single amino acid substitution in
sickle cell anemia
1. Translation of synthetic RNA
5’-UCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU-3’
N-SerLeuSerLeuSerLeuSerLeuSerLeu -C
N-LeuSerLeuSerLeuSerLeuSerLeuSer -C
H. Gobind Khorana
Nobel Prize, 1968
Combinations of alternating dinucleotides, trinucleotides
could be used to work out the code
2. Matching trinucleotides with amino acids
Nirenberg and Leder experiment
14C-Tyr
5’-UAC-3’
labeled ribosome
cell extract
ribosomes
14C-tRNATyr
3’
AUG5’
5’-UAC-3’
Ribosome-bound codon only binds to the corresponding tRNA
3. Point mutations leading to single amino acid substitutions
Charles Yanofsky, trp operon in E. coli
Single nucleotide change altered a single amino acid
Confirmed the code and showed it is not overlapping
4. Point mutation leading to a single amino acid substitution
in sickle cell anemia
Demonstrated the universality of the code and showed for the first time that
a genetic disease resulted from a change in a specific protein (gene)
The genetic code
Nearly universal across organisms – evolutionarily early
Degenerate – multiple codons for some amino acids (wobble)
Punctuation – start (AUG, Met) and stop (UAA, UGA, UAG)
Practice problem
Mix uracil and guanine to make mRNA
U:G=3:1
Synthesize protein in vitro
Analyze amino acid frequencies
Codon probabilities at U:G=3:1
codon
probability
ratio
UUU
P (UUU) = 3/4 X 3/4 X 3/4 = 27/64
P (UUG) = 3/4 X 3/4 X 1/4 = 9/64
P (UGU) = 3/4 X 1/4 X 3/4 = 9/64
P (GUU) = 1/4 X 3/4 X 3/4 = 9/64
P (UGG) = 3/4 X 1/4 X 1/4 = 3/64
P (GGU) = 1/4 X 1/4 X 3/4 = 3/64
P (GUG) = 1/4 X 3/4 X 1/4 = 3/64
P (GGG) = 1/4 X 1/4 X 1/4 = 1/64
0.42
UUG
UGU
GUU
UGG
GGU
GUG
GGG
0.14
0.14
0.14
0.05
0.05
0.05
0.01
total
1.00
Amino acid abundances at U:G=3:1
(UUU):(UUG):(UGU):(GUU):(UGG):(GGU):(GUG):(GGG)
0.42: 0.14: 0.14: 0.14: 0.05: 0.05: 0.05: 0.01
Phe : Leu : Cys : Val : Trp : Gly
0.42: 0.14 : 0.14: 0.19: 0.05: 0.06
Conclusions: Identify which codons specify which amino acids
Prove that the code is degenerate
tRNA – the final piece of the basic puzzle
Predicted by Francis Crick in 1955, published in 1958
“I cannot conceive of any structure (for either nucleic acid) acting as a direct
template for amino acids, or at least as a specific template………In its simplest
form there would be 20 different kinds of adaptor molecule, one for each amino
acid, and 20 different enzymes to join the amino acid to their adaptors.”
Identifed by Hoagland and Zamecnik in 1958
tRNAs specify the amino acid inserted
tRNATyr
Tyr
anticodon
3’
AUG5’
5’
NNNUACNNN3’
Tyr
codon
amino acyl tRNA synthetases load (“charge”) the tRNA with the amino acid
tRNAs and the suppressor concept
tRNATyr
WT
AUG GUG UAC GAC AAG AGA UAA
Met Val Tyr Asp Lys Arg Stop
tRNATyr SuTyr
Tyr
Mut 1 (Nonsense Mutant)
AUG GUG UAG GAC AAG AGA UAA
Met Val Stop
-
Mut 1 Su
AUG GUG UAG GAC AAG AGA UAA
Met Val Tyr Asp Lys Arg Stop
3’
AUG5’
5’
GUGUACGAC3’
Tyr
AUC5’
5’
GUGUAGGAC3’
Stop
Can you think of other types of suppressors?
3’
Practice problem
A protein found in E. coli has the following amino acid sequence:
Met-Leu-Trp-Ala-Ile-Ile-Cys-Asp
In a mutant strain of E. coli, the anticodon of a tRNA has been altered
from 5’-ACA-3’ to 5’-CCA-3’, resulting in a new amino acid sequence for
the protein. Predict the amino acid sequence of this new polypeptide.
Assume that the anticodon mutation alters the codon that the tRNA reads
but does not alter the amino acid that the tRNA is charged with.
cys
cys
anticodon (tRNA)
3’ACA 5’
3’ACC 5’
5’UGU 3’
5’UGG 3’
Cys
codon (mRNA)
Trp
Cys
3’
5’
ACA
5’
NNNUGUNNN3’
Cys
Met-Leu-Cys-Ala-Ile-Ile-Cys-Asp
3’
5’
ACC
5’
CUCUGGGCC3’
Cys
Lakota Sioux Native Americans believe the Goddess of
Peace once appeared in the form of a white buffalo calf.
As legend goes, chances are one in 10 million
that a white buffalo will ever enter this world.
Midterm
1. Happy to consider regrades if written in ink and submitted by 11/16/12
2. Exams have been photocopied
3. Final scores to grades for prior years (out of 405 total points)
A- or better
B- or better
C- or better
~350
~300
~200
4. We don’t know how the curve will turn out this year