Stepwise translocation of nucleic acid motors

Abstract

Recent single molecule studies have made a significant contribution to the understanding of the molecular mechanism involved in the movement of motor proteins which process DNA and RNA. Measurement of stepsize in two disparate motors, NS3 helicase and ribosome both revealed 3-bp steps, which consist of three hidden substeps. Combined with previous structural studies, NS3 is likely taking a single nucleotide step of translocation coupled to one ATP binding event and this mode may be conserved in multitude of helicases. Such a stepwise translocation movement appears to occur through main contacts with the phosphate backbone. Double stranded RNA and DNA motor, RIG-I and phi29, respectively, showed translocation on a duplex while tracking exclusively a single strand of RNA or DNA in a directional manner, 5'-3' in both cases. Spontaneous dynamics displayed by ribosome ratcheting and SSB (single stranded DNA binding protein) diffusing on DNA were rectified by interacting cofactors and proteins, EF-G and RecA, respectively.

Figure 1

A. NS3 unwinding was observed by single molecule FRET measurement where high FRET from two dyes placed at the duplex junction decreased stepwise during unwinding. Six steps obtained from 18 bp unwinding reveals 3 bp stepsize. B. Dwell time analysis revealed a nonexponential decay which indicates three hidden substeps within 3 bp unwinding steps. C. NS3 takes three steps of single nucleotide translocation coupled to ATP binding, which builds strain on the DNA-protein interface. The strain is released about every three basepairs, leading to a burst of 3bp unwinding observed in A.

Figure 2

A. NS3 and related helicases use two RecA like domains to translocate on the nucleic acid track, one nucleotide at a time in an inchworm like manner. The two domains ‘walk’ on the nucleic acid track by alternating affinities, mediated by ATP binding and ADP release. Dark and fair colored units represent tight and lose binding of two RecA like domains respectively. B. Two RecA like domains are depicted in the NS3 crystal structure (PDB: 1A1V) (9) with the same color designation as in A.

Figure 3

A. In the presence of EF-G elongation factor, the two rotating subunits of the ribosome translocate on mRNA taking 3 nucleotide steps corresponding to the size of one codon. B. The 30S and 50S subunits are represented on the ribosome structure. C. The ribosome undergoes a spontaneous ratcheting motion in the absence of EF-G.

Figure 4

A. NS3 crystallized without ATP shows two well conserved threonine residues (T269, T411 in blue) contacting the backbone phosphate 3 nts apart (PDB: 1A1V) (9). W501 (yellow), also known as “gatekeeper” is base stacked at the 3′ end of the bound nucleotide. B, C. Both Drosophilla Vasa (PDB:2DB3)(11) and eIF4AIII (PDB:2HYI) (10), two other SF2 helicases were co-crystallized with ATP analogs, AMPPNP and ADPNP, respectively. The structurally equivalent threonine residues (T375, T546 in Vasa, T164, T334 in eIF4IIIA) show phosphate contacts in 2 nts distance, suggesting 1 nt movement coupled to one ATP binding in between domain 1 and 2, bringing the two RecA-like domains together.