©1998 Timothy G. Standish Transcription and Translation Timothy G. Standish, Ph. D.
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©1998 Timothy G. Standish
Transcription Transcription and Translationand Translation
Timothy G. Standish, Ph. D.
©1998 Timothy G. Standish
Outline: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:The Genetic Code:A Brief A Brief
IntroductionIntroduction
DNA
mRNA
Transcription
IntroductionIntroduction
The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology
Cell
Polypeptide(protein)
TranslationRibosome
©1998 Timothy G. Standish
©1998 Timothy G. Standish
Information Only Goes One WayInformation Only Goes One WayThe 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 LanguageThe Genetic LanguageThe 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 LanguageThe Nucleotide Language
DNA-ATGCATGCATGCRNA-AUGCAUGCAUGCIt is not unlike different versions of
English.Me thinks it is a weaselI think it is a weasel
OH
OCH2
Sugar
HOH
A NucleotideA Nucleotide
NH2
N
N N
N
BaseP
O
OH
HO O
Phosphate
©1998 Timothy G. Standish
Pyrimidines
NH2
O
N
N NH
N
Guanine
N
N
Adenine
N
N
NH2
N O
NH2
N O
NH2
NCytosine
Uracil(RNA)CH3
N ON
O
NH
N ON
O
NH
Thymine(DNA)
Purines
©1998 Timothy G. Standish
Two Families of BasesTwo Families of Bases
©1998 Timothy G. Standish
Nucleotide WordsNucleotide 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.
SU
GA
R-P
HO
SP
HA
TE
BA
CK
BO
NE
B A
S E
S
H
PO
O
HO
O
O
CH2NH2N
NH
N
N
HOH
P
O
O
HO
O
O
CH2
NH2
N
N
N
N
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
A CodonA Codon
GuanineGuanine
AdenineAdenine
AdenineAdenine
Arginine
©1998 Timothy G. Standish
©1998 Timothy G. Standish
The Genetic CodeThe Genetic CodeHelps To Control Helps To Control
The Impact Of The Impact Of Point MutationsPoint Mutations
©1998 Timothy G. Standish
Redundancy in the CodeRedundancy 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 Why Not Use Shorter Codons?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
SentencesSentences 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 LanguageThe 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 LanguagesComparison of Languages
English - DogChinese -
DNA - CGTRNA - CGUAmino Acid -
Arginine
©1998 Timothy G. Standish
Redundancy:Redundancy:Synonyms and Codon DegeneracySynonyms 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
S E C O N D B A S E
A
GGUGGCGGAGGG
Gly*
AGUAGCAGAAGG
Arg
G
CGUCGCCGACGG
Arg
GUGUUGCUGAUGG
C
GAUGACGAAGAG
AAUAACAAAAAG
Glu
CAUCACCAACAG
AUAUUACUAAUAG
Stop
Tyr
GUUGUCGUAGUG
Val
AUUAUCAUAAUG start
Ile
CUUCUCCUACUG
Leu
UUUUUUCUUAUUG
Leu
Phe
Met/
GCUGCCGCAGCG
Ala
ACUACCACAACG
Thr
CCUCCCCCACCG
Pro
CUCUUCCUCAUCG
Ser
UCAG
U
UCAG
UCAG
UCAG
Gln†
His
Trp
Cys THIRD
BASE
FIRST
BASE
The Genetic CodeThe Genetic Code
Asp
Lys
Asn†
Stop
Ser
Neutral Non-polarPolarBasicAcidic
†Have aminegroups
*Listed as non-polar bysome texts
©1998 Timothy G. Standish
Codon AssignmentCodon AssignmentIs FortuitousIs 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 AssignmentCodon AssignmentIs FortuitousIs 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 CodeThe Genetic CodeIs Improbable And Is Improbable And
Does Not Look Does Not Look RandomRandom
©1998 Timothy G. Standish
Possible Codon AssignmentsPossible 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:
1
21
64
2.4 10 850.0000000000000000000000000
0000000000000000000000000000000000000000000000000000000000024
or
©1998 Timothy G. Standish
Problems With Codon Problems With Codon AssignmentAssignment
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 amion acids and “stop” must be assigned one codon, only 64 - 21 = 43 codons could be truely randomly assigned
Net probability is the likelyhood of initial assignment times probability of random assignment of remaining codons
1
21
1
64
1
21
43
1.0 10 60
©1998 Timothy G. Standish
Initial Codon AssignmentInitial Codon Assignment Theory would indicate initial codon assignment must have been
random Lewin in Genes VI pp 214, 215 suggests the following scenario:1 A small number of codons randomly get meanings representing a few
amino acids or possibly one codon representing a “group” of amino acids
2 More precise codon meaning evolves perhaps with only the first two bases having meaning with discrimination at the third position evolving later
3 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 AssignmentCodon AssignmentDoes not look randomDoes not look random
0
1
2
3
4
5
6
7
8
9
AminoAcids
1 2 3 4 5 6
Number of Codons
The genetic code does not like uneven numbers.
©1998 Timothy G. Standish
Initial Codon AssignmentInitial 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 AssignmentCodon AssignmentIs Not Strongly Correlated With UseIs Not Strongly Correlated With Use
Met
TrpCysHis
Tyr
Phe
ThrArg
Ser
Leu
Pro
Ile
GlnAsp
Lys
Glu
Asn
Val
GlyAla
1 2 3 4 5 6 Number of Codons
10
8
6
4
2
%In
Proteins
©1998 Timothy G. Standish
The Genetic CodeThe Genetic CodeIs Very Unlikely Is Very Unlikely
To ChangeTo Change
©1998 Timothy G. Standish
InitiationInitiation 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:How Codons Work:tRNA the TranslatorstRNA the Translators
tRNA - Transfer RNARelatively small RNA molecules that
fold in a complex way to produce a 3 dimensional shape with A helices
Associate a given amino acid with the codon on the mRNA that codes for it
©1998 Timothy G. Standish
AE
Large subunit
P
Small subunit
Translation - InitiationTranslation - Initiation
fMet
UACGAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
AE
Ribosome P UCU
Arg
Aminoacyl tRNA
PheLeu
Met
SerGly
Polypeptide
CCA
Translation - ElongationTranslation - Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
AE
Ribosome P
PheLeu
Met
SerGly
Polypeptide
Arg
Aminoacyl tRNA
UCUCCA
Translation - ElongationTranslation - Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
ANYTHING
ACIDAMINE
Protein SynthesisProtein Synthesis
C
O
OHCN
H
HH
C
HO H
C
H
O
CN
H
HH
C
H H
C
H
O
OHCN
H
HH
C
HO H
Serine
C
H
O
OHCN
H
HH
C
H H
AlanineH
C
O
OHC
R
N
H
H
Amino Acid
H2O
©1998 Timothy G. Standish
AE
Ribosome P
CCA
Arg
UCU
PheLeu
Met
SerGly
Polypeptide
Translation - ElongationTranslation - Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
AE
Ribosome P
Translation - ElongationTranslation - Elongation
Aminoacyl tRNA
CGA
Ala
CCA
Arg
UCU
PheLeu
Met
SerGly
Polypeptide
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
AE
Ribosome P
Translation - ElongationTranslation - Elongation
CCA
Arg
UCU
PheLeu
Met
SerGly
Polypeptide
CGA
Ala
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA5’mRNA
3’
©1998 Timothy G. Standish
Aminoacyl-tRNA SynthetaseAminoacyl-tRNA SynthetaseAminoacyl-tRNA Synthetase enzymes
attach the correct amino acids to the correct tRNA
This is an energy consuming processAminoacyl-tRNA Synthetases recognize
tRNAs on the basis of their looped structure, not by direct recognition of the anticodon
GlyAmino-acyl-tRNASynthetase
Gly
CCA
Amino-acyl-tRNASynthetase
GlyAmino-acyl-tRNASynthetase
AP
Amino-acyl-tRNASynthetase
MakingMakingAminoacyl-Aminoacyl-
tRNAtRNA
PPPyrophosphate
APAMP
Gly
CCA
Aminoacyl-tRNA
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.
AP
PP
ATP
©1998 Timothy G. Standish
©1998 Timothy G. Standish
Requirements for TranslationRequirements for Translation Ribosomes - rRNA and Protiens 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 CodonsReassignment 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 Changing Initial Codon AssignmentAssignment
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 CodeThe Genetic CodeIs Not Completely Is Not Completely
UniversalUniversal
©1998 Timothy G. Standish
Variation In Codon MeaningVariation 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
serine
Stop
Stop
Common Meaning
Stop
CandidaA yeast
Euplotes octacarinatusA ciliate
ParameciumA ciliate
OrganismTetrahymena thermophila
A ciliate
leucine
cysteine
glutamine
Modified Meaning
CUG
UGA
UAA UAG
Codon/sUAA UAG
glutamine
StopMycoplasma capricolumA bacteria tryptophanUGA
Neutral Non-polar, Polar
©1998 Timothy G. Standish
Variation in Mitochondrial Variation in Mitochondrial Codon AssignmentCodon Assignment
UGA/G=Stop
UniversalCode
Cyt
opla
sm/
Nu
cleu
s
Pla
nts
Yea
st/
Mol
ds
Pla
tyh
elm
ith
s
Ech
inod
erm
s
Mol
lusc
s
Inse
cts
Ver
teb
rate
s
UGA=Trp
AGA/G=Ser
AUA=Met
AUA=MetCUN=Thr
AUA=IleAAA=Asn
AAA=AsnN
emat
odes
NOTE - This would mean AUA changed from Ile to Met, then changed back to Ile in the Echinoderms
UGA must have changed to Trp then back to stop Differences in mtDNA lower the number of tRNAs needed
AAA must have changed from Lys to Asn twice
©1998 Timothy G. Standish
Summary: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 1Question 1 How many bases are in a codon?
A 1
B 2
C 3
D 4
E 5
©1998 Timothy G. Standish
Question 2Question 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 3Question 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
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