Co-Evolution ofthe Genetic Code and

Amino Acid BioSynthesis

Anna Battenhouse

An hypothesis from 1975by Jeffrey Tze-Fei Wong

Universal Phylogenetic Tree

Translation – the PlayersRibosome• large subunit: 23S rRNA,

many proteins– peptidyl transferase reaction,

tRNA sites

• small subunit: 16S rRNA, many proteins– messenger RNA (mRNA) contacts

Translation factors• EF-Tu, EF-G proteins, GTPtRNA (transfer RNA)

• acceptor arm holds amino acid• anticodon arm “reads” mRNA,

implements Genetic CodeaaRS (aminoacyl tRNA synthetase)

• “charge” tRNAs with the appropriate amino acid

22 “coded” amino acids

Chicken or Egg?

protein RNA

DNA

transcription

translation

synthesis,metabolism

excellent information storage,poor catalysis

poor information storage,excellent catalysis adequate information

storage,adequate catalysis

Simplifying Assumptions

• Ribosome proteins serve as scaffold• Small PTC RNA core with 2-fold symmetry

– A, P sites

• Translation factors not required– EF-Tu, EF-G, GTP

• “Proto-genes” were RNA molecules– copied by an RNA replicase ribozyme

• tRNA charging enzymes were ribozymes– left imprint on modern aaRSs

Science 256 (1992)

Benner, S.A., Ellington, A.D., Tauer, A., Modern metabolism as a palimpset of the RNA world PNAS 86 (1989)

The Pre-translation RNA world was metabolically complex

Diverse RNA enzymes (ribozymes), using cofactors and small random peptides

What’s Left to Explain?

What drove code evolution?

• Sterochemical interactions– Codon assignments arose from

Physical/chemical interactions between AAs and RNA

• Error minimization– Adjacency of codons minimizes potential

damage due to mutations/translation errors

• Expanding codons– Not all codon triplets used at first. Usage

expanded over time to modern 64.

• Amino acid biosynthesis– Formation/extension of AA biosynthetic

pathways

PNAS 55 (1966)

Woese et al., Microbio. Mol. Bio. Rev., 64:1 (2000)

7.5

9.1

7.5

9.1

Yarus 2009 Results

• RNA can bind wide variety of AAs specifically– polar, charged, aromatic– even aliphatic

• Several AA/RNA binding sites showed anticodon enrichment– Ile, Phe, Arg,

His, Trp, (Tyr)– However ~80% of triplets

not found

Woese, PNAS 55 (1966)

Direct RNA Template Model

Error Minimization

Amino Acid Biosynthesis Co-Evolution

Wong, J.T., Trends Bio. Sci.,

Feb. 1981

Wong, J.T., PNAS 73 (1976)

BioSynthesis Co-Evo Predictions

• AA biosynthesis is essential– phase 1 AA abundancy– phase 2 AA non-abundancy

• Biosynthetic evolutionary trace should still be discernable for precursor product pairs– codon allocation– “pre-translation” synthesis

• Set of encoded AAs is, in theory, (slightly) mutable

Not all amino acids would initially be available/abundant

Asn, Gln thermally unstable

Cys, Met, Trp, Phe, His

UV labile

Gly, Ala, Val, Leu Ile, Ser, Asp, Glu, initially most

abundant

Wong, J.T., Coevolution theory of the genetic code at age 30, BioEssays, 27.4 (2005)

Genetic Code by Biosynthetic Families

AAAA Precursor Product

Indirect Charging (“pre-translation”

biosynthesis)

Amino Acyl tRNA Synthetases (“aaRSs”) tRNA charging enzymes

AA AA

AA

AA

AA

Direct Charging

inventiveinventive biosynthesibiosynthesi

ss

Pre-translation Biosynthesis

Wong, J.T.,BioEssays 27.4 (2005)

Sep-tRNA Cys-tRNA(Sep = O-phosphoserine)

Lack of CysRS Euryarchaea

O’Donoghue et al.,PNAS 102:52 (2005)

Archaea

Archaea

Wong, J.T., Coevolution theory of the genetic code at age 30, BioEssays 27.4 (2005)

Distribution of Genes forPre-trans biosynthesis

Glu Gln Asp Asn

neither precursor nor product aaRSprecursor aaRS onlyboth precursor and product aaRS

Additional Evidence

• Phylogeny of aaRS genes– product aaRSs are often related to their

precursor aaRSs (and precursors more ancient)

• Enzyme for de novo Asn synthesis in many archaea was once an AspRS– pre-trans de novo biosynthesis via aaRS

paralog

• Natural and synthetic modifications to the Genetic code exist– pyrrolysine – 22nd amino acid– engineered AA additions in E. coli

Roy et al., and Francklyn, C., PNAS 100:17 (2003); Doring, et al., Scienece 292:501 (2001)

Pyrrolysine

• Incorporated in only a few prokaryotic proteins– has its own tRNA, (codon UAG, normally “stop”), aaRS

• Found in only a few species– Archaea

• 3 Methanosarcina• Methanococcoides

– Eubacteria• Desulfitobacterium hafniense (HGT)

• All species live off methylamine (fishy smell)– Pyl used in monomethylamine methyltransferase enzyme

Lehninger, Principles of Biochemistry, Fifth Ed.

Synthetic Code Expansion

BioSynth Co-Evo Theory Limitations

• Long on correlations, short on mechanisms• Does not address the important questions

surrounding tRNA– how did it arise? – did the anticodon arm develop independently

of the acceptor stem?– how did aaRSs come to be?

• and the Class I/Class II aaRS division

– role of the extensive AA base modifications

• What about the co-evolution of tRNAs and the 23S and 16S RNAs?– and the fascinating questions around message-

reading translocation

Blind men feeling an Elephant

Anticodon

wobble position

Acceptor stem

Transfer RNA (tRNA)

Maizels, N. et al., Biol. Bull. 196 (1999)

• Rossman fold active site• 2’ –OH attachment first• interacts with minor

groove of tRNA acceptor stem

• Beta sheet active site• 3’ –OH attachment• interacts with major

groove of tRNA acceptor stem

Schimmel et al., in The RNA World, Third Edition, Cold Spring Harbor Laboratory Press (2006)

Class I aaRSs Class II aaRSs

Giege, R. et al., Nucleic Acids Res. 26 (1998)

tRNA Identity Elements

Giege, R. et al., Nucleic Acids Res. 26 (1998)

Class I aaRS Class II aaRS

Xue, H., Tong, K., Marck, C., Grosjean, H., Wong, J.T., Transfer RNA paralogs, Gene 310 (2003)

tRNA phylogeny

Universal Phylogenetic Tree

Wobble

Watson/Crick A-U pair

Non-Watson/Crick G-U pair

I (inosine) can pair with C,U,A

Wobble Usage

Tong, K., Wong, J.T., Anticodon and wobble evolution, Gene 333 (2004)