On the origin of the genetic code Eörs Szathmáry

Eötvös University, Dept

of Plant Systematics,

Ecology and Theoretical

Biology

Centre for the

Conceptual Foundations

of Science, Munich

To the memory of Sergei Rodin

• Evolutionary scientist named to Susumu

Ohno Chair in Theoretical Biology

A major transition

• Novel way of using genetic information

• Division of labour between nucleic acids and proteins (replication, storage AND catalyis): molecular „germ” and „soma”

• Replicability and enzymatic function disturb each other

• Origin likely to comprise some idiosyncratic steps

Unambiguous and degenerate

The structure of the genetic code

• Amino acids in

the same

column of the

genetic code

are more

related to each

other physico-

chemically

Central nucleotide and amino acid

properties

Carl Woese

Constraints on codon reshuffling

for statistical investigations

Significance of some patterns

„The genetic

code is one in

a million” for

polarity

(Freeland and

Hurst)

Amino acid biosynthesis in E. coli

Biosynthetic relationships

Tzei-Fei Wong

Biosynthesis and amino acid chemistry

BOTH have shaped the code

• The code within the codons (Taylor &

Coates, 1989): first letter correlates with

biosynthesis, second letter with chemisty

• Szathmáry, E. & Zintzaras, E. (1992) A

statistical test of hypotheses on the

organization and origin of the genetic

code. J. Mol. Evol. 35, 185-189.

The RNA world may have preceded

the RNA-protein world

• Easy optimisation (with limits)

• Many artifical ribozymes (BUT no replicase)

• Coenzymes

• Ribozyme doing peptidyl transfer during protein

synthesis in ribosomes

• Amino acyl-tRNA synthetases are NOT the most

ancient proteins

• 20 residues are better than 4 in catalysis

Possibility for an experimental test

of the role of RNA stereochemistry

• Szathmáry, E. (1990) Towards the evolution of ribozymes. Nature 344, 115.

• Generate aptamers against different amino acids: see whether there is specific binding at all

• Search for codonic or anticodonic sequence accumulation in the binding sites

• Draw conclusions

Important ribozyme activities for the

emergence of translation

The fascinating work of Michael

Yarus

• Consistently carrying out

the research programme

for amino acid binding

aptamers

• Looking for increasingly

statistically significant

results

• Trying to put it into the

context of evolution

The minimal GCCU/GUGGC

ribozyme system

• The smallest ribozyme that

carries out a complex group

transfer is the sequence

GUGGC-3’,

• Acting to aminoacylate GCCU-

3’ (and host a manifold of

further reactions) in the

presence of substrate PheAMP.

Tryptophan-binding aptamers

The smallest typtophan binder

• The anticodon is CCA

• 13 fully conserved

nucleotides (26 bits of

information)

• Selective among

hydrophobic changes

The force of aptamer selection

(Yarus)

• Using recent sequences for 337 independent

binding sites directed to 8 amino acids and

containing 18,551 nucleotides in all, we show a

highly robust connection between amino acids and

cognate coding triplets within their RNA binding

sites.

• The apparent probability (P) that cognate triplets

around these sites are unrelated to binding sites is

congruent with 5.3 x 10(-45) for codons overall,

and P congruent with 2.1 x 10(-46) for cognate

anticodons.

Yarus aptamer codon/anticodon

table (2009)

Forces may have changed in strength

But what was the initial advantage?

• Evolution has no foresight

• Should confer some immediate advantage

• Concept of exaptation (preadaptation)

• Coded protein enzymes as culmination of a protracted phase of evolution

• Origin of the genetic code and protein synthesis are not necessariy the same thing

• Evolution is opportunistic

My reservation against uncoded

translation and statistical proteins

• Very wasteful process, initial selective advantage is unclear relative to the high cost of the machinery

• It is like proposing a scenario for the origin of language with long sentences but no meaning, where even word-meaning pairs are statistical!

• They usually completely ignore Yarus’s results!

Replicability and enzyme action are

in conflict

An independent catalytic alphabet is a cool

idea—provided you can get to it

Coding coenzyme handle (CCH)

hypothesis for the origin of the genetic

code (1990, 1993,…)

• This mechanism

works only if binding

between the kissing

hairpins follows the

umambiguous, but

degenerate principle of

the current genetic

code

Piecemeal vocabulary extension

• Amino acids are added and utilised one by

one

• No vicious error feedback as far as amino

acids are not involved (at the beginning) in

the functioning of synthetase ribozymes

• Coding precedes translation

Why indirect binding through base-

pairing? • N number of amino acids

• M number of metabolic enzymes

• If metabolic ribozymes specifically and directly bind amino acid cofactors, then 2 * M functionalities

• THE SITE FOR AMINO ACID BINDING WOULD BLOCK THE AA’S SPECIFIC GROUPS NO GOOD FOR CATALYSIS

• In contrast, only M specific synthetases are needed IF AMINO ACIDS ARE CHARGED TO SPECIFIC HANDLES!

• If M >> N, then choose synthetases

• Bind cofactors by their handles through base pairing (cheap)

Cofactor use by aptamers

CCH generates a prediction

• Missed previously

• Footprints of the evolution for catalytic

potential should be found in codon

clustering

• Kun et al. (2008) In: M. Barbieri (eds)

Codes of Life. Springer, Berlin.

Amino acid catalytic propensities

• Joint work with Kun, Pongor and Jordán (2008)

Significance of some patterns

Catalytic propensity and properties

Highest catalytic and β-turn

propensities

Substitution connectivity based on

the BLOSUM matrix

A minimalist enzyme

• Chorismate

mutase built

of 9 amino

acids only

Chiral histidine selection by D-

ribose RNA (Yarus) • The invariant choice of L-amino acids and D-ribose RNA for biological

translation requires explanation.

• Chiral choice using mixed, equimolar D-ribose RNAs having 15, 18,

21, 27, 35, and 45 contiguous randomized nucleotides was analyzed.

• These are used for simultaneous affinity selection of the smallest bound

and eluted RNAs using equal amounts of L- and D-His immobilized on

an achiral glass support, with racemic histidine elution.

• The most prevalent/smallest RNA sites are reproducibly and repeatedly

selected and there is a four- to sixfold greater abundance of L-histidine

sites. RNA’s chiral D-ribose therefore yields a more frequent fit to L-

histidine.

• Thus, if D-ribose RNA were first chosen biologically, translational L-

His usage could have followed.

Transfer RNAs with complementary anticodons:

Could they reflect early evolution of

discriminative genetic code adaptors? (1993)

• With regard to the anticodon loop and stem of pairs of

consensus tRNAs, complementary distances were

considerably less than direct distances-i.e., antiparallel

pairing invariably yielded fewer mismatches than direct

pairing.

• Each pair of pre-tRNAs with complementary anticodons

should have been almost identical with each other except for

their three central bases.

• The above situation appears to have dictated the early

establishment of direct links between anticodons and the

type of amino acids with which tRNAs are to be charged.

The presence of codon-anticodon

pairs in the acceptor stem of

tRNAs (1996)

• In pairs of consensus tRNAs with complementary

anticodons, their bases at the 2nd position of the

acceptor stem were also complementary.

• Accordingly, inverse complementarity was also

evident at the 71st position of the acceptor stem.

• The parallelism is especially impressive for the

pairs of tRNAs recognized by aminoacyl-tRNA

synthetases (aaRS) from the opposite classes.

Two types of aminoacyl-tRNA synthetases

could be originally encoded by

complementary strands of the same nucleic

acid (1995)

The growth of the

adaptor molecule

The first possible tetrad

ALL members of the first tetrad are

metabolically important

GLY

GCC

ALA

GGC

GUC

ASP

GAC

VAL

CU GA

• RNA synthesis (Gly,

Asp)

• Coenzyma A synthesis

(Val, Asp, Ala)

• Asp is also

catalytically important

Complementary anticodons and

parallel expansion into the catalytic

and structural worlds

• As a rule, pairs of complementary triplets encode

the functionally very different amino acids, most

often those with a high catalytic propensity (His,

Asp, Glu, Lys, Arg) contrasted with those with a

low catalytic but high structural (beta sheet

building) propensity (Val, Ile, Leu, Phe, Ala)

Second tetrad, catalytic expansion,

and the formation of the anticodon

loop?

ARG

GCG

ALA

CGC

GUG

HIS

CAC

VAL

CU GA

Experimentation with catalytic mini

RNAs (Yarus, 2010)

• The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2’-ribose hydroxyl using PheAMP.

• The initial 2’ Phe-RNA product can be elaborated into multiple peptidyl-RNAs.

Protein buildup on RNA scaffolds

• Shrinking RNA cores

• Selection for peptidyl-transferase activity

• Initially, proteins were strongly associated

with RNAs

• Could not fold by themselves

Proteins from pieces (Lupas,

2003)

Lupas’ conclusion

• The peptides forming these building blocks would

not in themselves have had the ability to fold, but

would have emerged as cofactors supporting RNA-

based replication and catalysis (the 'RNA world').

• Their association into larger structures and

eventual fusion into polypeptide chains would have

allowed them to become independent of their RNA

scaffold, leading to the evolution of a novel type of

macromolecule: the folded protein.

Ribosomal proteins cannot fold by

themselves (Lupas)

Thanks for your attention!

An ancient genetic code at the

anticodon?

• In eubacteria, a paralog of glutamyl-tRNA

synthetase, which lacks the tRNA-binding

domain, was found to aminoacylate tRNAAsp not

on the 30-hydroxyl group of the acceptor stem but

on a cyclopentene diol of the modified nucleoside

queuosine present at the wobble position of

anticodon loop.

• This modified nucleoside might be a relic of an

ancient code.

Amino acids in tRNA modifications

• At positions 34 and 37 of tRNA

Modified queuosine