Transcript Creation/Evolution - Geoscience Research Institute

Transcription
and Translation
Timothy G. Standish, Ph. D.
©1998 Timothy G. Standish
Outline:
The genetic code: A brief introduction
The genetic code helps control the impact of
point mutations
The genetic code is improbable and does
not look random
The genetic code is very unlikely to change
The genetic code is not completely
universal
Summary
©1998 Timothy G. Standish
The Genetic Code:
A Brief
Introduction
©1998 Timothy G. Standish
Introduction
The Central Dogma
of Molecular Biology
Cell
DNA
Transcription
Translation
mRNA
Ribosome
Polypeptide
(protein)
©1998 Timothy G. Standish
Information Only Goes One Way
The central dogma states that once “information”
has passed into protein it cannot get out again.
The transfer of information from nucleic acid
to nucleic acid, or from nucleic acid to protein,
may be possible, but transfer from protein to
protein, or from protein to nucleic acid, is
impossible. Information means here the precise
determination of sequence, either of bases in
the nucleic acid or of amino acid residues in
the protein.
Francis Crick, 1958
©1998 Timothy G. Standish
The Genetic Language
The genetic code is a written language
not unlike English or German.
While English uses 26 letters to spell
out words, genetic languages use only 4
nucleotide “letters”.
The nucleotide language of DNA is
transcribed into the nucleotide language
of RNA.
©1998 Timothy G. Standish
The Nucleotide Language
DNA-ATGCATGCATGC
RNA-AUGCAUGCAUGC
It is not unlike different versions
of English.
Me thinks it is a weasel
I think it is a weasel
©1998 Timothy G. Standish
A Nucleotide
Phosphate
HO
OH
P
O
Base
NH2
N
O
CH2
O
N
N
N
Sugar
OH
H
OH
©1998 Timothy G. Standish
Two Families of Bases
Purines
NH2
Adenine
N
N
N
Pyrimidines
O
CH3
N
O
N
Guanine
NH
N
(DNA)
NH2
O
Uracil
(RNA)
NH
N
N
Thymine
O
NH
N
O
NH2
Cytosine
N
N
O
©1998 Timothy G. Standish
Nucleotide Words
Words in the nucleotide
language are all 3 letters or
bases long.
These three base “words” are
called codons
This means that there can only
be 43 = 64 unique words.
©1998 Timothy G. Standish
A Codon
OH
P
HO
NH2
O
N
O
CH2
N
O
P
O
O
N
O
CH2
P
NH
N
O
Guanine
NH2
N
H
O
HO
N
H
O
HO
Adenine
N
NH2
O
N
O
CH2
O
OH
N
N
Adenine
Arginine
N
H
©1998 Timothy G. Stan
The Genetic Code
Helps To Control
The Impact Of
Point Mutations
©1998 Timothy G. Standish
Redundancy in the Code
Codons code for only 20 words, or amino
acids.
In addition to the amino acids, the start and
stop of a protein need to be coded for
There are thus a total of 22 unique
meanings for the 64 codons, so many
codons are synonyms.
The fact that many amino acids are coded
for by several codons is called degeneracy
©1998 Timothy G. Standish
Why Not Use Shorter
Codons?
If each codon was only 2 bases
long, there would be 42 = 16
possible unique codons
This would not provide enough
unique meanings to code for the 22
things (20 amino acids plus start
and stop) that have to be coded for.
©1998 Timothy G. Standish
Sentences
Sentences in the nucleic acid language are
called genes.
Each gene contains a sequence of codons
that describe the primary structure (amino
acid sequence) of a polypeptide (protein).
At the beginning of each gene is a start
codon
In the middle is a sequence of codons for
amino acids
At the end is a stop codon
©1998 Timothy G. Standish
The Protein Language
The protein language is very different from
the nucleotide language
Sentences are called polypeptides or
proteins
It is analogous to pictographic languages
like Chinese or Egyptian Hieroglyphics.
Each symbol has a meaning in pictographic
languages and in proteins, each amino acid
has a unique meaning or specific effect.
Words are not a sequence of nucleotides,
but each AA in the primary structure
©1998 Timothy G. Standish
Comparison of Languages
English - Dog
Chinese -
DNA - CGT
RNA - CGU
Amino Acid Arginine
©1998 Timothy G. Standish
Redundancy:
Synonyms and Codon Degeneracy
English - Synonyms
for dog:
Canine
Hound
Mutt
Cur
Pooch
Nucleic acids Synonyms for
Arginine:
CGU
CGC
CGA
CGG
AGA
AGG
©1998 Timothy G. Standish
The Genetic Code
Neutral Non-polar
Polar
Basic
Acidic
F
I U
R
S C
T
†Have amine
groups
*Listed as
non-polar by
some texts
B A
A
S G
E
SECOND
U
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
Phe
Leu
Leu
C
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
AUU
AUC Ile
AUA
AUGMet/start
ACU
ACC
ACA
ACG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
Val
BASE
A
Ser
UAU
UAC
UAA
UAG
Tyr
Pro
CAU
CAC
CAA
CAG
His
Thr
AAU
AAC
AAA
AAG
Asn†
Ala
GAU
GAC
GAA
GAG
Asp
Stop
Gln†
Lys
Glu
G
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
Cys
Stop
Trp
U
C
A
G
Arg
U
C
A
G
Ser
Arg
Gly*
U
C
A
G
U
C
A
G
T
H
I
R
D
B
A
S
E
©1998 Timothy G. Standish
Codon Assignment
Is Fortuitous
Effect of mutations is minimized in the
genetic code:
Mutation of the third base in a codon
changes the codon meaning only 1/3 of the
time
In AAs with only two codons, the mutation
always has to be purine to pyrimidine or
vice versa to change the AA coded for.
This is much harder than purine to purine or
pyrimidine to pyrimidine mutation
©1998 Timothy G. Standish
Codon Assignment
Is Fortuitous
Because of wobble base pairing, this
arrangement means less than 61 tRNAs have
to be made
53% of purine to purine or pyrimidine to
pyrimidine mutations in the second position
result in codons with either the same
meaning (i.e., UAA to UGA both = stop) or
coding for chemically related amino acids
©1998 Timothy G. Standish
The Genetic Code
Is Improbable And
Does Not Look
Random
©1998 Timothy G. Standish
Possible Codon Assignments
The probability of getting the assignment of codons to
amino acids we have can be calculated as follows:
– There are 21 meanings for codons:
20 amino acids
1 stop
1 start, which doesn’t count because it also is assigned to methionine
– 64 Codons
If we say that each codon has an equal probability of
being assigned to an amino acid, then the probability of
getting any particular set of 64 assignments is:
64
 1 
85
or

2.4

10
21
0.0000000000000000000000000
0000000000000000000000000
0000000000000000000000000
00000000024
©1998 Timothy G. Standish
Problems With Codon
Assignment
Under Miller-Urey type conditions, more than the 20
amino acids would have been available
To estimate probability, we assume only 20, but this
changes the odds
As all 20 amino acids and “stop” must be assigned one
codon, only 64 - 21 = 43 codons could be truly
randomly assigned
Net probability is the likelihood of initial assignment
times probability of random assignment of remaining
43
codons
 1  1  1 
60
 1.0  10


216421
©1998 Timothy G. Standish
Initial Codon Assignment
1
2
3
Theory would indicate initial codon assignment must
have been random
Lewin in Genes VI, p 214, 215 suggests the following
scenario:
A small number of codons randomly get meanings
representing a few amino acids or possibly one codon
representing a “group” of amino acids
More precise codon meaning evolves perhaps with only
the first two bases having meaning with discrimination at
the third position evolving later
The code becomes “frozen” when the system becomes so
complex that changes in codon meaning would disrupt
existing vital proteins
©1998 Timothy G. Standish
Codon Assignment
Does not look random
9
8
7
6
Amino 5
Acids 4
3
2
1
0
1
2
3
4
5
Number of Codons
6
The genetic code does not like uneven numbers.
©1998 Timothy G. Standish
Initial Codon Assignment
If natural selection worked on codons,
the most commonly used amino acids
might be expected to have the most
codons
If there was some sort of random
assignment, the same thing might be
expected
This is not the case
©1998 Timothy G. Standish
Codon Assignment
Is Not Strongly Correlated With Use
10
Leu
8
Glu
%
In 6
Proteins
Lys
Asp
Gln
Asn
Phe
4
Ile
Ala
Gly
Ser
Val
Thr
Pro
Arg
Tyr
2
Met
His
Cys
Trp
1
2
3
4
Number of Codons
5
6
©1998 Timothy G. Standish
The Genetic Code
Is Very Unlikely
To Change
©1998 Timothy G. Standish
Initiation
The small ribosome subunit binds to the 5’
untranslated region of mRNA
The small ribosomal subunit slides along the
mRNA 5’ to 3’ until it finds a start codon
(AUG)
The initiator tRNA with methionine binds to
the start codon
The large ribosomal subunit binds with the
initiator tRNA in the P site
©1998 Timothy G. Standish
How Codons Work:
tRNA the Translators
tRNA - Transfer RNA
Relatively small RNA molecules that
fold in a complex way to produce a 3dimensional shape with A helices
Associate a given amino acid with the
codon on the mRNA that codes for it
©1998 Timothy G. Standish
Translation - Initiation
fMet
Large
subunit
E
P
A
UAC
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
Small mRNA
subunit
©1998 Timothy G. Standish
Translation - Elongation
Polypeptide
Arg
Met
Phe
Leu
Ser
Aminoacyl tRNA
Gly
Ribosome
E
P
A
CCA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©1998 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Aminoacyl tRNA
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©1998 Timothy G. Standish
Protein Synthesis
AMINE H
H
O
N
ACID
C
C
ANYTHING
R
H
Amino Acid
Alanine
OH
H
H
Serine
H
O
N
C
OH
H
C
H
H
H
H2O
H
H
C
H
C
H
O
C
N
OH
C
HO
N
H
O
C
C
C
H
H C
H
H HO
H
OH
C
H
H
N
H
H
C
O
©1998 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©1998 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ala
Ser
Gly
Aminoacyl tRNA
Arg
Ribosome
E
P
A
CCA
UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©1998 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
Ala
P
A
UCU CGA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©1998 Timothy G. Standish
Aminoacyl-tRNA Synthetase
Aminoacyl-tRNA Synthetase enzymes
attach the correct amino acids to the
correct tRNA
This is an energy-consuming process
Aminoacyl-tRNA Synthetases
recognize tRNAs on the basis of their
looped structure, not by direct
recognition of the anticodon
©1998 Timothy G. Standish
Gly
P
P
Making
AminoacyltRNA
Aminoacyl-tRNA
Synthetase
P
ATP
Gly
P
P
P
Aminoacyl-tRNA
Synthetase
Pyrophosphate
Gly
Aminoacyl-tRNA
Synthetase
Gly
Aminoacyl-tRNA
Synthetase
P
AMP
CCA
AminoacyltRNA
CCA
Note that the amino acid is not paired with the
tRNA on the basis of the anticodon. The correct
tRNA for a given amino acid is recognized on
the basis of other parts of
the molecule.
©1998 Timothy G. Standish
Requirements for Translation
Ribosomes - rRNA and Proteins
mRNA - Nucleotides
tRNA
– The RNA world theory might explain these three
components
Aminoacyl-tRNA Synthetase
– A protein, thus a product of translation and cannot be
explained away by the RNA world theory
L Amino Acids
ATP - For energy
This appears to be an irreducibly complex system
©1998 Timothy G. Standish
Reassignment of Stop Codons
Changes in stop codon meaning must have occurred after
meanings were “frozen” in other organisms, alternatively
organisms that exhibit them must have evolved from
organisms that never shared the universal genetic code
All changes in stop codons must include three changes:
– Replacement of stop codons that do not code for stop anymore
with those that still do
– Production of new tRNAs with anticodons that recognize the
codon as not stop anymore
– Modification of the release factor (eRF) to restrict its binding
specificity further so that it no longer binds the stop codon with
new meaning
All changes “appear to have occurred independently in
specific lines of evolution” (Lewin, Genes VI)
©1998 Timothy G. Standish
Changing Initial Codon
Assignment
Once codons have been assigned to an amino
acid, changing their meaning would require:
– Changing the tRNA anticodon or, much harder,
changing the aminoacyl-tRNA synthetase
– Changing all codons to be reassigned in at least the
vital positions in those proteins needed for survival
This seems unlikely
The situation is complicated in cases where
genes seem to have been swapped between the
nucleus and mitochondria
©1998 Timothy G. Standish
The Genetic Code
Is Not Completely
Universal
©1998 Timothy G. Standish
Variation In Codon Meaning
Lack of variation in codon meanings across almost all phyla is
taken as an indicator that initial assignment must have occurred
early during evolution and all organisms must have descended
from just one individual with the current codon assignments
Exceptions to the universal code are known in a few single-celled
eukaryotes and mitochondria and at least one prokaryote
Most exceptions are modifications of the stop codons UAA, UAG
and UGA
Organism
Codon/s
Tetrahymena thermophila UAA UAG
A ciliate
Paramecium
UAA UAG
A ciliate
Common Meaning Modified Meaning
Stop
glutamine
Stop
glutamine
Euplotes octacarinatus
UGA
Stop
cysteine
Mycoplasma capricolum
UGA
Stop
tryptophan
Candida
CUG
serine
leucine
A ciliate
A bacteria
A yeast
Neutral Non-polar, Polar
©1998 Timothy G. Standish
AUA=Met
CUN=Thr
Universal
Code
AAA=Asn
AUA=Ile
AAA=Asn
Vertebrates
Insects
Molluscs
Echinoderms
Nematodes
Platyhelmiths
Yeast/
Molds
Plants
Cytoplasm/
Nucleus
Variation in Mitochondrial
Codon Assignment
UGA/G=Stop
NOTE - This would mean
AUA changed from Ile to
Met, then changed back to
AUA=Met
Ile in the Echinoderms
AGA/G=Ser
AAA must have changed from Lys to
Asn twice
UGA=Trp
UGA must have changed to Trp then back to stop
Differences in mtDNA lower the number of tRNAs needed
©1998 Timothy G. Standish
Summary
The genetic code appears to be non-random
in nature and incorporates considerable
safeguards against harmful point mutations
An evolutionary model suggests at least at
some level of randomness in assignment of
amino acids to codons
No mechanism exists for genetic code
evolution
Thus variation in the genetic code suggests a
polyphyletic origin for life
©1998 Timothy G. Standish
Question 1
How many bases are in a codon?
A 1
B 2
C 3
D 4
E 5
©1998 Timothy G. Standish
Question 2
True or False
A) Mutating just one base in a codon may have a
profound effect on the protein being coded for and
consequently the organism
B) Mutating the third base in a codon frequently has
no effect on the protein being coded for
C) Changing an amino acid in a protein will have less
effect on a protein if the amino acid belongs to the
same class as the original amino acid it is replacing
©1998 Timothy G. Standish
Question 3
Which of the following components of the translation
process cannot be explained away by the RNA World
theory?
A) mRNA
B) Ribosomes
C) Aminoacyl-tRNA transferase
D) tRNA
©1998 Timothy G. Standish