Experiments and theory about an elementary coding system based on RNA

Brookhaven Laboratory 01/13/2008

Jean LehmannCenter 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

G0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP aa-RNA + AMP

3) Translation: peptide-RNA1 + aa-RNA2

G0 < 0 RNA1 + (peptide + 1)-RNA2

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, establisha 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, establisha translation system compatible with them

http://www.uic.edu/classes/bios/bios100/mike/spring2003/lect04.htm

Small RNAstem-loop

Folding of small random RNA sequences

Lehmann et al., 2004. J. theor. Biol. 227:381-395

(nucleotides)

Illangasekare and Yarus 1999. RNA 5, 1482-1489

Self-aminoacylating ribozymes

size: 29 nucleotides

NaCl 100 mMMgCl2 80 mMCaCl2 40 mM0ºC pH 7.0

k2nd

Aminoacylation mechanism

(modern tRNA)

3’ extension

~ 25 nucleotides ~ 75 nucleotides

HPLC analysisof ribozyme activity

Mass spectroscopy

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f product =1− exp(k2nd A0

kVal −AMP

(exp(−kVal −AMP t) −1))

3’ extensions :GUUACG (squares)GUUUUACG (triangles)GUUUUUUACG (circles)

Kinetics of aminoacylation

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d[ribozyme]

dt= −k2nd [ribozyme]A0 exp(−kVal −AMP t)

Solution:

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A0 = [val − AMP]0

Lehmann et al., 2007.RNA 13:1191-1197

Variant 3’ extension (5’-3’) fproduct (t = 30 minutes)

(%)

krel

V1 GUCG 1.7 1

V2 GUUCG 37.6 28

V3 GUUGACG 56.0 48

O riginal 29-m er GUUAUGACG 61.8 56

V4 GUUAUUGACG 47.4 38

V5 (poly-U) GUUUUUUACG 89.5 131

V6 GUUAUGAUCG 1.7 1

V7 GUUAUGAUUCG 8.1 5

V8 GUUAUUUGACG 48.8 39

V9 GUUAGAUUUCG 17.3 11

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krel =k2nd (a)

k2nd (b)=

ln 1− f product(a ) t( )( )

ln 1− f product(b ) t( )( )

Influence of the bases in the extension

Lehmann et al., 2007. RNA 13:1191-1197

(the smallest ribozyme)

Lehmann et al. in prep.

1) Activation of the amino acid (aa): aa + ATP aa-AMP + ppi

G0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP aa-RNA + AMP

3) Translation: peptide-RNA1 + aa-RNA2

G0 < 0 RNA1 + (peptide + 1)-RNA2

existing ribozymes

Extending the catalytic repertoireof self-aminoacylating ribozymes

1) Activation of the amino acid (aa): aa + ATP aa-AMP + ppi

G0 ≈ 02) Esterification of the tRNA: RNA + aa-AMP aa-RNA + AMP

3) Translation: peptide-RNA1 + aa-RNA2

G0 < 0 RNA1 + (peptide + 1)-RNA2

wanted ribozyme

Extending the catalytic repertoireof self-aminoacylating ribozymes

Original ribozymePossible form ofthe 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, establisha 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, establisha 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, 2000. J. theor. Biol 202:129-144

codons

Influence of neighboringgroups on the rateof a chemical reaction

Lightstone and Bruice, 1996J. Am. Chem. Soc.118, 2595

Analytical model for an elementary translation:Two amino acids, two anticodon-codon couples

Parametersa = kcat(2)/kcat(1)

b = kcat(1)/k–(1)

c = k–(1)/k–(2)

d = [aa(2)]/[aa(1)]k+(1) = k+(2)

a, b, c, d > 0

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p 11( ) = [aa(1)]k+(1)

kcat(1)

k−(1) + kcat(1)

[aa(1)]k+(1)

kcat(1)

k−(1) + kcat(1)

+ [aa( 2)]k+(1)

kcat(2)

k−(1) + kcat(2)

⎛

⎝ ⎜

⎞

⎠ ⎟

amino acid codon

k+

k–(2)

a prioriconfigurations

translation

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coding Σ := p 11( ) = p 2 2( ) >1 2 Two parameters fixed:a = 100 c = 0.01

coding phenomenon observed if b ~ 1

Our analysis provides some answersto the origin of the correlation (and the code!)

Parametersinvolved:b, c

Parameters involved:a, b, d

Lehmann, in prep.

two codons,two amino acids:

~80%~20%

~20%~80%

a ~ 100b ~ 1c ~ 0.01d ~ 0.1

Our analysis provides some answersto the origin of the correlation (and the code!)

Parametersinvolved:b, c

Parameters involved:a, b, d

Lehmann, in prep.

two codons,two amino acids:

~80%~20%

~20%~80%

a ~ 100b ~ 1c ~ 0.01d ~ 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 YeAxel Buguin

Hanna SalmanCarine DouarcheYusuke 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 proteinon the organism

Selection

protein = DPcode ( I )

Selection = f (protein) = f ° DPcode ( 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, 1449-1459

Activation step as catalyzed by a Synthetase(amino acid = glycine)

Arnez et al., 1999 J. Mol. Biol 286, 1449-1459

glycine ATP

Activation step as catalyzed by a Synthetase(amino acid = glycine)

Arnez et al., 1999 J. Mol. Biol 286, 1449-1459

glycine-AMP ppi

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ξ(n) ≈ pentropy ⋅ pbending = δn−3 2 exp(−λlp

na)

Probability that the end of the extension lies onthe binding site within a small interval of time t:

: parametersa : length monomerlp : persistence lengthn : nb monomers

Poly-U in the extension: effect of the length

Miller - prebiotic synthesis experiment