Inter-Subunit Coordination in a Homomeric Ring-ATPase

Homomeric ring ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the direct observation of the intersubunit coordination and step size of such a ring ATPase, the double-stranded-DNA packaging motor in the bacteriophage ϕ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 base pairs (bp). Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated by means of a mechanism novel for ring ATPases. Furthermore, a step size that is a non-integer number of base pairs demands new models for motor–DNA interactions.

[1]  Paul J Jardine,et al.  Bacteriophage phi 29 DNA packaging. , 2002, Advances in virus research.

[2]  S. Mukherjee,et al.  DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 DNA helicase. , 2006, Molecular cell.

[3]  G. Oster,et al.  Reverse engineering a protein: the mechanochemistry of ATP synthase. , 2000, Biochimica et biophysica acta.

[4]  Peixuan Guo,et al.  Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. , 1987, Journal of molecular biology.

[5]  S. Grimes,et al.  The bacteriophage phi29 packaging proteins supercoil the DNA ends. , 1997, Journal of molecular biology.

[6]  H. Flyvbjerg,et al.  Power spectrum analysis for optical tweezers , 2004 .

[7]  Steven M. Block,et al.  Analysis of high resolution recordings of motor movement. , 1995, Biophysical journal.

[8]  Derek N. Fuller,et al.  Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage phi29. , 2008, Biophysical journal.

[9]  L. Joshua-Tor,et al.  Mechanism of DNA translocation in a replicative hexameric helicase , 2006, Nature.

[10]  M. Schnitzer,et al.  Statistical kinetics of processive enzymes. , 1995, Cold Spring Harbor symposia on quantitative biology.

[11]  A. Wilkinson,et al.  AAA+ superfamily ATPases: common structure–diverse function , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[12]  J. Berger,et al.  Evolutionary relationships and structural mechanisms of AAA+ proteins. , 2006, Annual review of biophysics and biomolecular structure.

[13]  Paul R. Selvin,et al.  Single-molecule techniques : a laboratory manual , 2008 .

[14]  H. Gutfreund,et al.  Enzyme kinetics , 1975, Nature.

[15]  M. Emmett,et al.  Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states , 2005, Nature Structural &Molecular Biology.

[16]  R. Cross,et al.  Mechanics of the kinesin step , 2005, Nature.

[17]  M. Latterich,et al.  The AAA team: related ATPases with diverse functions. , 1998, Trends in cell biology.

[18]  Dwight L. Anderson,et al.  Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29. , 2001, Journal of structural biology.

[19]  J. Berger,et al.  Structural Insights into RNA-Dependent Ring Closure and ATPase Activation by the Rho Termination Factor , 2006, Cell.

[20]  Carlos Bustamante,et al.  Exact solutions for kinetic models of macromolecular dynamics. , 2008, The journal of physical chemistry. B.

[21]  G. Oster,et al.  Mechanochemistry of transcription termination factor Rho. , 2006, Molecular cell.

[22]  Tania A. Baker,et al.  Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines , 2005, Nature.

[23]  E. Parzen On Estimation of a Probability Density Function and Mode , 1962 .

[24]  M. Rossmann,et al.  Defining molecular and domain boundaries in the bacteriophage phi29 DNA packaging motor. , 2008, Structure.

[25]  J. Berger,et al.  Structure of the Rho Transcription Terminator Mechanism of mRNA Recognition and Helicase Loading , 2003, Cell.

[26]  S. Tans,et al.  The bacteriophage straight phi29 portal motor can package DNA against a large internal force. , 2001, Nature.

[27]  Hiroyasu Itoh,et al.  Rotation of F1-ATPase: how an ATP-driven molecular machine may work. , 2004, Annual review of biophysics and biomolecular structure.

[28]  E. Mancini,et al.  Hexameric molecular motors: P4 packaging ATPase unravels the mechanism , 2006, Cellular and Molecular Life Sciences CMLS.

[29]  G. Oster,et al.  Mechanochemistry of t7 DNA helicase. , 2005, Journal of molecular biology.

[30]  S. Grimes,et al.  Bacteriophage φ29 DNA packaging , 2002 .

[31]  Jan Löwe,et al.  Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. , 2006, Molecular cell.

[32]  D. Gai,et al.  Mechanisms of Conformational Change for a Replicative Hexameric Helicase of SV40 Large Tumor Antigen , 2004, Cell.

[33]  Michael R Sawaya,et al.  Crystal Structure of T7 Gene 4 Ring Helicase Indicates a Mechanism for Sequential Hydrolysis of Nucleotides , 2000, Cell.

[34]  J. Volff Gene and protein evolution. Preface. , 2007, Genome dynamics.

[35]  Marc C. Morais,et al.  Structure of the bacteriophage φ29 DNA packaging motor , 2000, Nature.

[36]  C. Chen,et al.  Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation , 1997, Journal of virology.

[37]  Detlef D. Leipe,et al.  Evolutionary history and higher order classification of AAA+ ATPases. , 2004, Journal of structural biology.

[38]  L. Kellner The Near Infra-Red Absorption Spectrum of Heavy Water , 1937 .

[39]  Jens Michaelis,et al.  Mechanism of Force Generation of a Viral DNA Packaging Motor , 2005, Cell.

[40]  S. Grimes,et al.  A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi 29. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Dickerson,et al.  Analysis of local helix geometry in three B-DNA decamers and eight dodecamers. , 1991, Journal of molecular biology.

[42]  Derek N. Fuller,et al.  Ionic effects on viral DNA packaging and portal motor function in bacteriophage φ29 , 2007, Proceedings of the National Academy of Sciences.

[43]  E. Mancini,et al.  Atomic Snapshots of an RNA Packaging Motor Reveal Conformational Changes Linking ATP Hydrolysis to RNA Translocation , 2004, Cell.

[44]  Eugene V Koonin,et al.  Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging. , 2004, Nucleic acids research.

[45]  Carlos Bustamante,et al.  Recent advances in optical tweezers. , 2008, Annual review of biochemistry.

[46]  Carlos Bustamante,et al.  Supplemental data for : The Bacteriophage ø 29 Portal Motor can Package DNA Against a Large Internal Force , 2001 .

[47]  Carlos Bustamante,et al.  Differential detection of dual traps improves the spatial resolution of optical tweezers. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Nancy R Forde,et al.  Mechanical processes in biochemistry. , 2004, Annual review of biochemistry.

[49]  L. Aravind,et al.  Comparative genomics and evolutionary trajectories of viral ATP dependent DNA-packaging systems. , 2007, Genome dynamics.

[50]  Z. Koza Maximal force exerted by a molecular motor. , 2002, Physical review. E, Statistical, nonlinear, and soft matter physics.

[51]  S. Smith,et al.  Ionic effects on the elasticity of single DNA molecules. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[52]  Alan R. Lowe,et al.  ATPase site architecture and helicase mechanism of an archaeal MCM. , 2007, Molecular cell.