Evolution of the Genetic Code:Before and After the LUCA

1. The genetic code evolved to its canonical form before the Last Universal Common Ancestor of Archaea, Bacteria and Eukaryotes - >3 billion years ago. It appears to be highly optimized. How did it get to be this way?

2. Numerous small changes have occurred to the canonical code since then. What is the mechanism of codon reassignment?

Codon Reassignment – The Genetic code is variable in mitochondria (and also some cases of other types of genomes)

Second Position

U C A G Third Pos.

FirstPosition

U

F FLL

SS SS

Y YStopStop

C CStop W

U C A G

C

L LLL

PP PP

H HQ Q

RR RR

U C A G

A I II M

TT TT

N NK K

S SRR

U C A G

G VV V V

AA AA

D DE E

GG GG

U C A G

UGA Stop to Trp

AUA Ile to Met

CUN Leu to Thr

CGN Arg to unassigned

AGR Arg to Ser to Stop/Gly

etc.....

But how can this happen? It should be disadvantageous.

Porifera

Cnidaria

Arthropoda

Nematoda

Platyhelminthes

Lophotrochozoa

Echinodermata

Hemichordata

Urochordata

Cephalochordata

Craniata

AAA : Lys -> Asn

Loss of tRNA-Ile(CAU) but AUA remains Ile

Loss of tRNA-Arg(UCU) and AGR : Arg -> Ser

AGR : Ser -> Stop

AGR : Ser -> Gly

AUA : Ile -> Met

Loss of many tRNAs + import from cytoplasm

AAA : Lys -> unassigned

Reassignments in Metazoa

Example 1: AUA was reassigned from Ile to Met during the early evolution of the mitochondrial genome.

Before Codon Anticodon Notes

Ile

Ile

Ile

Met

AUU

AUC

AUA

AUG

GAU

k2CAU

CAU

G in the wobble position of the tRNA-Ile can pair with U and C in the third codon position Bacteria and some protist mitochondria possess another tRNA-Ile with a modified base that translates AUA only.

The tRNA-Met translates AUG only.

After Codon Anticodon Notes

Ile

Ile

Met

Met

AUU

AUC

AUA

AUG

GAU

UAU or

f5CAU

In animal mitochondria the k2CAU tRNA has been deleted.

There is a gain of function of the tRNA-Met by a mutation or a base modification

Example 2: UGA was reassigned from Stop to Trp many times (12 times in mitochondria).

Before Codon Anticodon Notes

Stop

Trp

UGA

UGG

RF

CCA

Release Factor recognizes UGA codon.

Normal tRNA-Trp translates only UGG codons.

After Codon Anticodon Notes

Trp

Trp

UGA

UGG

UCA In animal mitochondria (and elsewhere) there is a gain of function of the tRNA-Trp via mutation or base modification so that it translates both UGG and UGA.

The GAIN-LOSS framework

(Sengupta & Higgs, Genetics 2005)

LOSS = deletion or loss of function of a tRNA or RF

GAIN = gain of a new tRNA or a gain of function of an existing one.

Mutations in coding sequences

Initial Code.No Problem.

Ambiguous codon.Selective disadvantage.

Unassigned codon.Selective disadvantage.

New Code.Selective disadvantage because codons are used in wrong places

GAIN

GAINLOSS

LOSS

New Code.Codons now used in right places.No Problem.

Note – the strength of the selective disadvantage depends on the number of times the codon is used. There is no disadvantage if the codon disappears.

Four possible mechanisms of codon reassignment.

1. Codon Disappearance - The codon disappears. The order of the gain and loss is irrelevant.

For the other three mechanisms the codon does not disappear.

2. Ambiguous Intermediate – The gain happens before the loss. There is a period when the gain is fixed in the population and translation is ambiguous.

3. Unassigned Codon – The loss happens before the gain. There is a period when the loss is fixed in the population and the codon is unassigned.

4. Compensatory Change – The gain and loss are fixed in the population simultaneously (although they do not arise at the same time). There is no intermediate period between the old and the new codes. - cf. theory of compensatory substitutions in RNA helices.

Sengupta & Higgs (2005) showed that all four mechanisms work in a population genetics simulation

Codon reassignment

No. of

times

Can this be explained by

GCAU mutation pressure?

Change in No.

of tRNAs

Is mispairing important?

Mechanism

UAG: Stop Leu 2 G A at 3rd pos. +1 No CD

UAG: Stop Ala 1 G A at 3rd pos. +1 No CD

UGA: Stop Trp12

G A at 2nd pos. 0

Possibly. CA at 3rd pos.

CD

CUN: Leu Thr 1 C U at 1st pos. 0 No CD

CGN: Arg Unass 5 C A at 1st pos. -1 No CD

AUA: Ile Met or Unassigned

3 / 5No

-1Yes. GA at 3rd pos.

UC

AAA: Lys Asn 2

No0

Yes. GA at 3rd pos.

AI

AAA: Lys Unass1

No0

Possibly. GA at 3rd pos.

UC or AI

AGR: Arg Ser 1

No-1

Yes. GA at 3rd pos.

UC

AGR: Ser Stop 1 No 0 No AI(b)

AGR: Ser Gly 1 No +1 No AI(b)

UUA: Leu Stop 1 No 0 No UC or AI

UCA: Ser Stop 1 No 0 No UC or AI

Summary of Codon Reassignments in Mitochondria

CD mechanism explains disappearance of stop codons because they are rare initially. Only a few examples of CD for sense codons. UC and AI are important for sense codons.

Three examples in yeasts (Mutation pressure GC to AU)

Second Position

U C A G Third Pos.

FirstPosition

U

F FLL

SS SS

Y YStopStop

C CStop W

U C A G

C

L LLL

PP PP

H HQ Q

RR RR

U C A G

A I II M

TT TT

N NK K

S SRR

U C A G

G VV V V

AA AA

D DE E

GG GG

U C A G

CGN is rare (replaced by AGR)

CGN Arg codons become unassigned.

CUN is rare (replaced by UUR)

CUN Leu to Thr

AUA and AUU common and AUC is rare

Nevertheless AUA is reassigned to Met. Codon does not disappear

LeuCUN

Leu UUR

Arg CGN

Arg AGR

S 53 192 7 33

Y. 44 618 0** 75

C 3 279 12 29

C 132 397 47 26

C 66 547 39 45

P 25 714 18 67

K 0 286 0** 48

C 11* 294 1** 45

S 33* 333 7 49

S 19* 274 0** 40

S 22* 300 0** 46

Leu and Arg codons in yeasts

Codon Disappearance causes reassignments

* CUN = Thr. Unusual tRNA-Thr present instead of tRNA-Leu

** CGN = unassigned. tRNA-Arg is deleted

AUU AUC AUA AUG AUA is tRNAJ 133 40 32 48 Ile K2CAUO 161 34 0 57 Absent noneP 113 39 49 51 Ile K2CAU

Codon UsageAUA Ile to Met in Yeasts

AUU AUC AUA AUGC 119 81 229 100 Ile K2CAUC 303 32 193 117 Ile K2CAUP 274 18 562 105 Ile K2CAUK 213 16 7 63 ? noneC 207 21 16 73 Met C*AUS 239 31 60 73 Met C*AUS 203 7 101 56 Met C*AUS 218 11 95 70 Met C*AU

codon anticodon

AUU Ile GUA

AUC Ile “

AUA Ile K2CAU

AUG Met CAU

Evolution of the canonical code - Before the LUCA

The canonical code seems to be optimized to reduce the effects of translational and mutational errors.

Neighbouring codons code for similar amino acids.

5 7 9 11 13

C LI F WMY V PT A HQSG NKR E D

Woese’s polar requirement scale

Measure difference between amino acid properties by how far apart they are on this scale.

Principal Component Analysis Projects the 8-d space into the two ‘most important’ dimensions.

Big

Small

Hydrophobic Hydrophilic

Cost function g(a,b) for replacing amino acid a by amino acid b

e.g. difference in Polar Requirement

i j

iji j

jiij raagrE /),(

rij = rate of mistaking codon i for codon j

= 1 for single position mistakes, 0 otherwise

E = measure of error associated with a code

Generate random codes by permuting the 20 amino acids in the code table

E is smaller for the canonical code than for almost all random codes.

E

p(E)Ereal

f

f ~ 10-6

one in a million codes is better (Freeland and Hurst)

GAU

GAC

GAA

GAG

AAU

AAC

AAA

AAG

CAU

CAC

CAA

CAG

UAU

UAC

UAA

UAG

Asp

Asp

Gu

Asn

Ays

Lys

His

Hn

Gln

Tyr

Tyr

*

*

GCU

GCC

GCA

GCG

ACU

ACC

ACA

ACG

CCU

CCC

CCA

CCG

UCU

UCC

UCA

UCG

Ala

Ala

Ala

Ala

Ser

Ser

Ser

Ser

GUU

GUC

GUA

GUG

AUU

AUC

AUA

AUG

CUU

CUC

CUA

CUG

UUU

UUC

UUA

UUG

Val

Val

Val

Val

Ile

Ile

Ile

Met

Leu

Leu

eu

Phe

Phe

Leu

Leu

GGU

GGC

GGA

GGG

AGU

AGC

AGA

AGG

Ser

Ser

Arg

Arg

CGU

CGC

CGA

CGG

Arg

Ag

UGU

UGC

UGA

UGG

Cp

Pro

Pro

Pro

Pro

The statistical argument shows that the code is highly non-random but it does not explain how the code evolved to be that way. Need a step-by-step evolutionary argument that leads from a proposed first stage of the code to today’s code.

Random permutations – Not Possible

Random swaps – seems unlikely

The earliest code probably had few amino acids. Which were the first? Selection acts when new amino acids are added.

GAU

GAC

GAA

GAG

AAU

AAC

AAA

AAG

CAU

CAC

CAA

CAG

UAU

UAC

UAA

UAG

Asp

Asp

Gu

Asn

Ays

Lys

His

Hn

Gln

Tyr

Tyr

*

*

GCU

GCC

GCA

GCG

ACU

ACC

ACA

ACG

CCU

CCC

CCA

CCG

UCU

UCC

UCA

UCG

Ala

Ala

Ala

Ala

Ser

Ser

Ser

Ser

GUU

GUC

GUA

GUG

AUU

AUC

AUA

AUG

CUU

CUC

CUA

CUG

UUU

UUC

UUA

UUG

Val

Val

Val

Val

Ile

Ile

Ile

Met

Leu

Leu

eu

Phe

Phe

Leu

Leu

GGU

GGC

GGA

GGG

AGU

AGC

AGA

AGG

Ser

Ser

Arg

Arg

CGU

CGC

CGA

CGG

Arg

Ag

UGU

UGC

UGA

UGG

Cp

Pro

Pro

Pro

Pro

Time scale for the origin of life

The origin of the genetic code is the end of the RNA World

What preceded RNA? Another polymer? Metabolism only?

Dating of rocks and meteorites

Last ocean- vaporizing impact. Lunar craters

Isotopic evidence for life

Microfossil evidence

Stromatolites.

Phylogenetic methods (divergence after LUCA)

Prebiotic synthesis of organic molecules Miller-Urey experiment (1953)

Began with a mixture of CH4 , NH3, H2O and H2.Energy source = electric spark or UV light.Obtained 10 amino acids.

Atmospheres and Chemistry

reducing: CH4 , NH3, H2O, H2. or CO2, N2, H2 or CO, N2, H2 There is hydrogen gas and/or hydrogen is present combined with other elements (methane, ammonia, water)

neutral: CO or CO2 , N2 , H2O

no hydrogen or oxygen gas

oxidizing: O2, CO2, N2

oxygen gas present

Prebiotic chemists favour reducing atmospheres.

Yields in Miller-Urey exp are higher and more diverse in reducing than in neutral atmospheres. Doesn’t work in oxidizing atmosphere.

Planetary AtmospheresMajor element in universe is H (big bang) so doesn’t it make sense that atmosphere was reducing?Jupiter retains original mixture: H2, He + small amounts CH4, NH3, H2O

Smaller planets lose H2

New atmosphere created by outgassing from interior

Geologists & Astronomers favour an intermediate atmosphere.

(i) Venus - 64 Earth atmospheres pressure! Mostly CO2 and N2

(ii) Carbonates in sedimentary rocks on Earth suggest previously lots of CO2

Current Earth: Mostly N2, O2 + small amounts of CO2 H2O – changed by life.

Mars: very low pressure – mostly CO2 and N2

So maybe Miller and Urey were wrong? :-(

Alternative suggestion – Hydrothermal vents

Sea water passes through vents.

Heated to 350o C. Cools to 2o C in surrounding ocean.

Supply of H2 H2S etc.

Fierce debate as to whether these conditions favour formation or breakup of organic molecules (Miller & Lazcano, 1995)

Organic compounds in meteorites

Most widely studied meteorite is the Murchison meteorite. Fell in Australia in 1969. Carbonaceous chondrite.

Contained both biological and non-biological amino acids

Both optical isomers (later shown to be not quite equal)

Compounds are not contamination

Just about all the building block molecules have now been found in carbonaceous meteorites (Sephton, 2002).

Astrochemistry: molecular clouds; icy grains; parent bodies of meteorites....

Delivery by: dust particles; meteorites; comets....

Was external delivery an important source of organic molecules?

The earliest code probably had few amino acids. Which were the first? Selection acts when new amino acids are added.

GAU

GAC

GAA

GAG

AAU

AAC

AAA

AAG

CAU

CAC

CAA

CAG

UAU

UAC

UAA

UAG

Asp

Asp

Gu

Asn

Ays

Lys

His

Hn

Gln

Tyr

Tyr

*

*

GCU

GCC

GCA

GCG

ACU

ACC

ACA

ACG

CCU

CCC

CCA

CCG

UCU

UCC

UCA

UCG

Ala

Ala

Ala

Ala

Ser

Ser

Ser

Ser

GUU

GUC

GUA

GUG

AUU

AUC

AUA

AUG

CUU

CUC

CUA

CUG

UUU

UUC

UUA

UUG

Val

Val

Val

Val

Ile

Ile

Ile

Met

Leu

Leu

eu

Phe

Phe

Leu

Leu

GGU

GGC

GGA

GGG

AGU

AGC

AGA

AGG

Ser

Ser

Arg

Arg

CGU

CGC

CGA

CGG

Arg

Ag

UGU

UGC

UGA

UGG

Cp

Pro

Pro

Pro

Pro

Prebiotic Synthesis of amino acids

Higgs and Pudritz (2009) Astrobiology

Amino acids are found in

• Meteorites

• Atmospheric chemistry experiments (Miller-Urey)

• Hydrothermal synthesis

• Icy dust grains in space

Rank amino acids in order of decreasing frequency in 12 observations. Derive ranking.

Miller Murchison Yamato Ice Exp.Gly 1.000 1.00 1.000 1.000Ala 1.795 0.34 0.380 0.293Asp 0.077 0.19 0.035 0.022Glu 0.018 0.40 0.110Val 0.044 0.19 0.100 0.012Ser 0.011 0.003 0.072Ile 0.011 0.13 0.060Leu 0.026 0.04 0.035Pro 0.003 0.29 0.001Thr 0.002 0.003

Comparison of amino acid frequencies produced non-biologically

10 amino acids are found in the Miller-Urey experiments. Very similar ones are also found in meteorites, an Ice grain analogue experiment, and other places.

These are ‘early’ amino acids that were available for use by the first organisms.

G A D E V S I L P T

The other 10 are not seen. These are late amino acids that were only used when organisms evolved a means of synthesizing them biochemically.

K R H F Q N Y W C M

concentrations normalized

relative to Gly

The earliest amino acids are those that are cheapest to form thermodynamically

Positions of early and late amino acids....

What does this mean?

Second Position

U C A G Third Pos.

FirstPosition

U

F FLL

SS SS

Y YStopStop

C CStop W

U C A G

C

L LLL

PP PP

H HQ Q

RR RR

U C A G

A I II M

TT TT

N NK K

S SRR

U C A G

G VV V V

AA AA

D DE E

GG GG

U C A G

M

FF

Maybe only 2nd position was relevant initially.

Late amino acids took over codons previously assigned to amino acids with similar properties.

U C A G

U

Val Ala Asp Gly

UC

AG

CUC

AG

AUC

AG

GUC

AG

Propose that the four earliest amino acids were Val, Ala, Asp, Gly

Four column code. (Higgs Biol. Direct. 2009)

This is a triplet code but only the second base means anything.

The second base is the most important for codon-anticodon recognition.Unlikely to make a mistake at second position.

All first and third position mistakes are synonymous.

Code structure after addition of the 10 early amino acids..

Add new amino acids in positions that were formerly occupied by amino acids with similar properties. This minimizes disruption to existing gene sequences.

Summary of my argument -

Selection acts at the time of addition of new amino acids to the code. The new amino acid is assigned to codons that formerly coded for an amino acid with similar properties. This minimizes disruption to existing genes.

The result is that codons in the same columns end up assigned to amino acids with similar properties. The column structure is retained from the earliest code.

Hence the code appears to minimize translational error with respect to randomly reshuffled codes, even though translational error was not the main factor being selected.

Pathways of amino acid synthesis in modern organisms (from Di Giulio 2008)

Other points –

Column structure suggests that translational errors were more important than mutational errors (tRNA structure/RNA world)

Precursor-product pairs tend to be neighbours (but doubts over statistical significance). Maybe late amino acids took over codons previously assigned to their biochemical precursors.

Direct chemical interactions between RNA motifs and amino acids (“stereochemical theory”). In vitro selection experiments suggest binding sites of aptamers preferentially contain codon and anticodon sequences.

RNA WorldFirst hypothesis:

There was a stage of evolution at when RNA molecules performed both genetic and catalytic roles. DNA later took over the genetic role and proteins took over the catalytic role.

Translation depends on RNA:mRNA supplies the information for protein synthesis.Active ingredient of the ribosome is rRNA – 3d structures show site of peptidyl transferase reaction. Proteins probably added as a late addition to the ribosome.tRNAs also essential for translation.

Second hypothesis:

The RNA world arose de novo in the form of self replicating ribozymes.

Almost certainly true

The jury is still out

Self-splicing introns. First RNA catalysts to be discovered. Tom Cech (1982).

‘RNA World’ term coined by Walter Gilbert (1986).

RNA world idea originated in 60’s as a theoretical solution to the chicken and egg problem of DNA and proteins.

Hammerhead ribozyme

Cleaves RNA at a specific point.

Rolling circle mechanism of replication of virus-like RNAs in plants. Chops long strand into pieces.

Example of an RNA catalyst

What can ribozymes do? Ligases

T. A. Lincoln, G. F. Joyce, Science 323, 1229 (2009)

EBA E’

An Autocatalytic Set Made from Ligases

T. A. Lincoln, G. F. Joyce, Self-Sustained Replication of an RNA Enzyme, Science 323, 1229, (2009)

Given a supply of A, B, A’, B’, the E and E’ make more of themselves.

EBA E ' ''' EBA E

What can ribozymes do?Recombinases

E.J. Hayden, G.v. Kiedrowski & N. Lehman, Angew. Chem. Int. Edit. (2008) 120, 8552

Catalyst is autocatalytic given a supply of W X Y Z.The non-covalent assembly is also a catalyst.

What can ribozymes do?Polymerases

Black +Blue – ribozymeRed – templateOrange – primer

Primer extended by up to 14 nucleotides

Johnstone et al. (2001) Science

Gradual improvement of Polymerases in the lab

Wochner et al. (2011) Science - up to 95 nucleotides

What can ribozymes do?Nucleotide Synthetases

Unrau and Bartel, (1998) Nature

An RNA organism must have had a metabolism.

Hypothetical pathway for RNA catalyzed RNA synthesis (Joyce)

Synthesis of nucleosides

Phosphorylation

Generation of NTPs

Creation of activated nucleotides

Stepwise polymerization

Clutter of RNA synthesis (Joyce)

Why is this particular set of monomers used for nucleic acids?

How is this set synthesized specifically?

Where is the chemistry occurring? Earth, or space? Hydrothermal vents?

MW Powner et al. Nature 459, 239-242 (2009) doi:10.1038/nature08013

Previously assumed synthesis of -ribocytidine-2',3'-cyclic phosphate 1 (blue; note the failure of the step in which cytosine 3 and ribose 4 are proposed to condense together) and the successful new synthesis described here (green). p, pyranose; f, furanose.

A new route to Pyrimidine ribonucleotide assembly.

Chemical synthesis of monomers and polymers must have occurred before the origin of ribozymes.

Ferris (2002) Orig. Life Evol. Biosph.Montmorillonite catalyzed synthesis of RNA oligonucleotides (30-50 mers)

Rajamani et al. (2008) Orig. Life Evol. Biosph.Lipid assisted synthesis of RNA-like polymers from mononucleotides

Costanzo et al. (2009) J. Biol. Chem.Synthesis of long RNA strands from cyclic nucleotides in water

Rajamani et al. (2010) J. Am. Chem. Soc.Measurements of error rates in non-enzymatic RNA replication

There are still some experimental issues…But this is a logical necessity!

How could the RNA world have got started?

Getting from chemistry to biology….

RNA replicators must have emerged from prebiotic synthesis of random sequences

Monomers

Activatedmonomers

Shortpolymers

Long polymers

PrecursorsSynthesis

Polymerization

Activation

Polymerization

Ribozymes

catalyze catalyze

catalyze

catalyze

Jump-starting the RNA WorldWu & Higgs (2009) J. Mol. Evol.

RNA

Are there alternatives to RNA?

a – Threose Nucleic Acid – TNA c – Glycerol derived nucleic acidb – Peptide nucleic acid – PNA d – Pyranosyl RNA

RNA hybridizes with other nucleic acids. Information is not lost.

DNA-RNA hybrids DNA takes over at end of RNA world.

Maybe TNA or PNA preceded the RNA world. Information passed to RNA.

Would need to show that the alternative was easier to synthesize than RNA.

Two scenarios from Segré & Lancet (2000)

A – RNA first (strong RNA world hypothesis)

B – Lipids first (lipid world hypothesis – compositional genomes – metabolism without genes)