Development of single-molecule techniques and their application to biophysical questions
Keywords: Applied Physics.

This dissertation covers three research projects, the instrumentation needed to conduct them, and the physics which underlie the techniques. All projects employ optical tweezers as a primary method, but are otherwise quite diverse: two are strictly in vitro experiments, while one includes extensive computer simulation; two involve protein, while the third considers a nucleic acid system; two employ single-molecule FRET to measure an additional reaction coordinate, while one gains its data solely from a single-beam optical trap. Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde IFT, ferrying cargo along MTs toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by the absolute numbers of each motor type on a given train, how prone KIF3AB is to dissociation from MTs relative to KIF17, and how prone both motors are to dissociation relative to binding MTs. The TPP riboswitch is a cis-regulatory element in mRNAs that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and FRET, using an optical tweezers instrument equipped for simultaneous single-molecule FRET. The "force-FRET" approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in single-molecule FRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity. Hairpins in nascent RNAs extend RNAP pause durations, but the mechanism of stabilization is poorly understood. It was speculated that a conformational change in the RNAP clamp induced by the formation of the hairpin in the RNA exit channel was responsible. Using a dual-beam optical tweezers instrument with single-molecule FRET detection capabilities, we examine the conformation of the clamp during elongation, elemental pauses, hairpin-stabilized pauses, and termination pauses. We find that there is only negligible change in the clamp conformation upon entry to a hairpin-stabilized pause, with a displacement an order of magnitude small than had been predicted. Based on our and prior results, we speculate as to other possible mechanisms. In the course of this work, I developed a full suite of applications to efficiently control and collect data from a modern optical tweezers instrument, which adheres to best practices for realtime data collection; it can now be freely downloaded