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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
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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.

Dec 26, 2015

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Page 1: 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.

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

Page 2: 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 genetic code

Lehmann 2006 Springer Verlag

Page 3: 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 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

Page 4: 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.

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.

Page 5: 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.

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

Page 6: 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.

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

Page 7: 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.

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

Small RNAstem-loop

Page 8: 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.

Folding of small random RNA sequences

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

(nucleotides)

Page 9: 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.

Illangasekare and Yarus 1999. RNA 5, 1482-1489

Self-aminoacylating ribozymes

size: 29 nucleotides

Page 10: 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.

NaCl 100 mMMgCl2 80 mMCaCl2 40 mM0ºC pH 7.0

k2nd

Aminoacylation mechanism

(modern tRNA)

3’ extension

~ 25 nucleotides ~ 75 nucleotides

Page 11: 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.

HPLC analysisof ribozyme activity

Mass spectroscopy

Page 12: 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.

f product =1− exp(k2nd A0

kVal −AMP

(exp(−kVal −AMP t) −1))

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

Kinetics of aminoacylation

d[ribozyme]

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

Solution:

A0 = [val − AMP]0

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

Page 13: 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.

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

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

Page 14: 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 smallest ribozyme)

Lehmann et al. in prep.

Page 15: 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.

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

Page 16: 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.

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

Page 17: 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.

Original ribozymePossible form ofthe wanted ribozyme

Page 18: 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.

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

Page 19: 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.

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

Page 20: 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 genetic code

Lehmann 2006 Springer Verlag

Page 21: 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.

A correlation in the genetic code:physico-chemical constraints at the level of translation

Lehmann, 2000. J. theor. Biol 202:129-144

codons

Page 22: 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.

Influence of neighboringgroups on the rateof a chemical reaction

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

Page 23: 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.

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

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

Page 24: 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.

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

coding phenomenon observed if b ~ 1

Page 25: 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.

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

Page 26: 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.

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

Page 27: 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.

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.

Page 28: 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.

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)

Page 29: 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.
Page 30: 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.

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.

Page 31: 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.

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

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

Page 32: 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.

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

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

glycine ATP

Page 33: 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.

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

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

glycine-AMP ppi

Page 34: 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.

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

Page 35: 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.

Miller - prebiotic synthesis experiment