Kinetic measurement of ribosome motor stalling force

We measure the ribosome motor stalling forces to unzip mRNA polymers during gene expression. An approach of using the changes in the reaction rate constants to determine the molecular motor forces is presented. Specific antisense DNA oligomers complementary to mRNA templates are used as kinetic barriers for estimating the ribosome forces using real time bioluminescence detection of luciferase gene expression. The rate constants are determined by comparing the experimental data with numerical simulation of gene expression to deduce the ribosome force (26.5±1pN) required to unzip mRNA polymers. Understanding the forces generated by the ribosome may also enable the construction of information-based artificial nanoscale machines.

[1]  Christoph F. Schmidt,et al.  Direct observation of kinesin stepping by optical trapping interferometry , 1993, Nature.

[2]  Upinder S Bhalla,et al.  Understanding complex signaling networks through models and metaphors. , 2003, Progress in biophysics and molecular biology.

[3]  Development of single-molecule tracking confocal microscope combined with force spectroscopy for gene-expression analysis , 2004 .

[4]  Simona Cocco,et al.  Force and kinetic barriers to unzipping of the DNA double helix , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[5]  G. I. Bell.  Models for the specific adhesion of cells to cells. , 1978, Science.

[6]  Nancy R. Forde,et al.  Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  V. Ramakrishnan,et al.  Ribosome Structure and the Mechanism of Translation , 2002, Cell.

[8]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[9]  M. Rief,et al.  Sequence-dependent mechanics of single DNA molecules , 1999, Nature Structural Biology.

[10]  M. Kozak,et al.  New Ways of Initiating Translation in Eukaryotes? , 2001, Molecular and Cellular Biology.

[11]  A Maritan,et al.  Dynamical scaling of the DNA unzipping transition. , 2001, Physical review letters.

[12]  T. Maniatis,et al.  An extensive network of coupling among gene expression machines , 2002, Nature.

[13]  J. Liphardt,et al.  Reversible Unfolding of Single RNA Molecules by Mechanical Force , 2001, Science.

[14]  N. Sugimoto,et al.  Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. , 1995, Biochemistry.

[15]  U. Bockelmann,et al.  Mechanical separation of the complementary strands of DNA. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  E. Evans.  Probing the relation between force--lifetime--and chemistry in single molecular bonds. , 2001, Annual review of biophysics and biomolecular structure.

[17]  M. Zuker.  Calculating nucleic acid secondary structure. , 2000, Current opinion in structural biology.

[18]  T. Polenova,et al.  KINETICS OF BIOLUMINESCENCE IN THE FIREFLY LUCIFERIN LUCIFERASE SYSTEM , 1994 .

[19]  Michelle D. Wang,et al.  Force and velocity measured for single molecules of RNA polymerase. , 1998, Science.

[20]  J. Spudich,et al.  Single myosin molecule mechanics: piconewton forces and nanometre steps , 1994, Nature.