©1998 Timothy G. Standish Transcription and Translation Timothy G. Standish, Ph. D.

©1998 Timothy G. Standish

Transcription Transcription and Translationand Translation

Timothy G. Standish, Ph. D.


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


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



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


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.


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


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


Two Families of BasesTwo Families of Bases


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



The Genetic CodeThe Genetic CodeHelps To Control Helps To Control

The Impact Of The Impact Of Point MutationsPoint Mutations


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


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.


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


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


Comparison of LanguagesComparison of Languages

English - DogChinese -

DNA - CGTRNA - CGUAmino Acid -

Arginine


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


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


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


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


The Genetic CodeThe Genetic CodeIs Improbable And Is Improbable And

Does Not Look Does Not Look RandomRandom


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


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


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


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.


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


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


The Genetic CodeThe Genetic CodeIs Very Unlikely Is Very Unlikely

To ChangeTo Change


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


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


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’


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’


AE

Ribosome P

PheLeu

Met

SerGly

Polypeptide

Arg

Aminoacyl tRNA

UCUCCA



3’


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


AE

Ribosome P

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide



3’


AE

Ribosome P


Aminoacyl tRNA

CGA

Ala

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide


3’


AE

Ribosome P


CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide

CGA

Ala


3’


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



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


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)


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


The Genetic CodeThe Genetic CodeIs Not Completely Is Not Completely

UniversalUniversal


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


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


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


Question 1Question 1 How many bases are in a codon?

A 1

B 2

C 3

D 4

E 5


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


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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©1998 Timothy G. Standish Transcription and Translation Timothy G. Standish, Ph. D.

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