Experiments and theory about an elementary coding system based on RNA Brookhaven Laboratory 01/13/2008 Jean Lehmann Center for Studies in Physics and Biology The Rockefeller University, New York

The genetic code Lehmann 2006 Springer Verlag

The three chemical reactions required for translation: 1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppi  G 0 ≈ 0 2) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP 3) Translation: peptide-RNA 1 + aa-RNA 2   G 0 < 0 RNA 1 + (peptide + 1)-RNA 2 existing ribozymes

Research goals The main goal of this research is to establish a minimal form of the translation process based on small RNA structures, without proteins. It is expected that a simplified genetic code will be associated with this polymerization process. Theoretical challenge: Make a bridge between the laws of kinetics and thermodynamics, relevant to describe the events at the molecular level, and a theory of coding.

Major Steps of the research 1) Understand the structural requirements for an RNA to load an amino acid without enzyme 2) Once these RNAs will be isolated, establish a translation system compatible with them

Major Steps of the research 1) Understand the structural requirements for an RNA to load an amino acid without enzyme 2) Once these RNAs will be isolated, establish a translation system compatible with them

Small RNA stem-loop

Folding of small random RNA sequences Lehmann et al., J. theor. Biol. 227: (nucleotides)

Illangasekare and Yarus RNA 5, Self-aminoacylating ribozymes size: 29 nucleotides

NaCl 100 mM MgCl 2 80 mM CaCl 2 40 mM 0ºC pH 7.0 k 2nd Aminoacylation mechanism (modern tRNA ) 3’ extension ~ 25 nucleotides~ 75 nucleotides

HPLC analysis of ribozyme activity Mass spectroscopy

3’ extensions : GUUACG (squares) GUUUUACG (triangles) GUUUUUUACG (circles) Kinetics of aminoacylation Solution: Lehmann et al., RNA 13:

Influence of the bases in the extension Lehmann et al., RNA 13:

(the smallest ribozyme) Lehmann et al. in prep.

1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppi  G 0 ≈ 0 2) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP 3) Translation: peptide-RNA 1 + aa-RNA 2   G 0 < 0 RNA 1 + (peptide + 1)-RNA 2 existing ribozymes Extending the catalytic repertoire of self-aminoacylating ribozymes

1) Activation of the amino acid (aa): aa + ATP  aa-AMP + ppi  G 0 ≈ 0 2) Esterification of the tRNA: RNA + aa-AMP  aa-RNA + AMP 3) Translation: peptide-RNA 1 + aa-RNA 2   G 0 < 0 RNA 1 + (peptide + 1)-RNA 2 wanted ribozyme Extending the catalytic repertoire of self-aminoacylating ribozymes

Original ribozyme Possible form of the wanted ribozyme

Major Steps of the research 1) Understand the structural requirements for an RNA to load an amino acid without enzyme 2) Once these RNAs will be isolated, establish a translation system compatible with them

Major Steps of the research 1) Understand the structural requirements for an RNA to load an amino acid without enzyme 2) Once these RNAs will be isolated, establish a translation system compatible with them

The genetic code Lehmann 2006 Springer Verlag

A correlation in the genetic code: physico-chemical constraints at the level of translation Lehmann, J. theor. Biol 202: codons

Influence of neighboring groups on the rate of a chemical reaction Lightstone and Bruice, 1996 J. Am. Chem. Soc.118, 2595

Analytical model for an elementary translation: Two amino acids, two anticodon-codon couples amino acidcodon k+k+ k –(2) a priori configurations translation

Two parameters fixed: a = 100 c = 0.01 coding phenomenon observed if b ~ 1

Our analysis provides some answers to the origin of the correlation (and the code!) Parameters involved: b, c Parameters involved: a, b, d Lehmann, in prep. two codons, two amino acids: ~80% ~20% ~80% a ~ 100 b ~ 1 c ~ 0.01 d ~ 0.1

Our analysis provides some answers to the origin of the correlation (and the code!) Parameters involved: b, c Parameters involved: a, b, d Lehmann, in prep. two codons, two amino acids: ~80% ~20% ~80% a ~ 100 b ~ 1 c ~ 0.01 d ~ 0.1

Conclusions and outlook Part 1: We now better understand the structural features enabling small RNAs to covalently attach (activated) amino acids (reaction 2). The coupling between activation (reaction 1) and aminoacylation (reaction 2) still needs to be demonstrated. Part 2: A correlation shows that the crossing of the last chemical step leading to polymerization (reaction 3) is conditioned by physico-chemical constraints. The establishment of a translation system compatible with the ribozymes studied in Part 1 still needs to be demonstrated.

Acknowledgements: Albert Libchaber Shixin Ye Axel Buguin Hanna Salman Carine Douarche Yusuke Maeda Funding: Swiss National Science foundation (Fellowships I & II ) The Rockefeller University (M.J & H. Kravis Fellowship)

Piece of information ( I ) (DNA or RNA) Phenotype (protein) Biological Evolution decoding process DP -code- Effect of the protein on the organism  Selection protein = DP code ( I ) Selection = f (protein) = f ° DP code ( I ) If the code is not unique, there are as many possible selections on a given I as there are ways of decoding DP. Selection cannot operate at the same time on the information and on the code. The original code is therefore not the product of selection.

Activation step as catalyzed by a Synthetase (amino acid = glycine) Arnez et al., 1999 J. Mol. Biol 286,

Activation step as catalyzed by a Synthetase (amino acid = glycine) Arnez et al., 1999 J. Mol. Biol 286, glycineATP

Activation step as catalyzed by a Synthetase (amino acid = glycine) Arnez et al., 1999 J. Mol. Biol 286, glycine-AMPppi

Probability that the end of the extension lies on the binding site within a small interval of time  t:  : parameters a : length monomer l p : persistence length n : nb monomers Poly-U in the extension: effect of the length

Miller - prebiotic synthesis experiment