Binding and translocation of termination factor rho studied at the single-molecule level.

Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5'→3' toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.

Precision steering of an optical trap by electro-optic deflection.

We designed, constructed, and tested a single-beam optical trapping instrument employing twin electro-optic deflectors (EODs) to steer the trap in the specimen plane. Compared with traditional instruments based on acousto-optic deflectors (AODs), EOD-based traps offer a significant improvement in light throughput and a reduction in deflection-angle (pointing) errors. These attributes impart improved force and position resolution, making EOD-based traps a promising alternative for high-precision nanomechanical measurements of biomaterials.

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.

The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated ("gated") and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the single-molecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.

Statistical kinetics of macromolecular dynamics.

Fluctuations in biochemical processes can provide insights into the underlying kinetics beyond what can be gleaned from studies of average rates alone. Historically, analysis of fluctuating transmembrane currents supplied information about ion channel conductance states and lifetimes before single-channel recording techniques emerged. More recently, fluctuation analysis has helped to define mechanochemical pathways and coupling ratios for the motor protein kinesin as well as to probe the contributions of static and dynamic disorder to the kinetics of single enzymes. As growing numbers of assays are developed for enzymatic or folding behaviors of single macromolecules, the range of applications for fluctuation analysis increases. To evaluate specific biochemical models against experimental data, one needs to predict analytically the distribution of times required for completion of each reaction pathway. Unfortunately, using traditional methods, such calculations can be challenging for pathways of even modest complexity. Here, we derive an exact expression for the distribution of completion times for an arbitrary pathway with a finite number of states, using a recursive method to solve algebraically for the appropriate moment-generating function. To facilitate comparisons with experiments on processive motor proteins, we develop a theoretical formalism for the randomness parameter, a dimensionless measure of the variance in motor output. We derive the randomness for motors that take steps of variable sizes or that move on heterogeneous substrates, and then discuss possible applications to enzymes such as RNA polymerase, which transcribes varying DNA sequences, and to myosin V and cytoplasmic dynein, which may advance by variable increments.