Our primary research
project is an extension of Professor G. Holzwarth's work: the
measurements of the drag force and mechanical work required for
fast transport of vesicles and the relationship of this cellular task to
the known limitations of motor proteins, especially kinesin. In buffer,
against the force of an optical trap, the maximum of steady force
which kinesin can exert is 6.5 pN. One ATP is hydrolyzed per 8 nm
step, and each step takes 50 microseconds. About 100 such steps
occur per second during processive movement. In a cell, the
vesicle(load) is in cytoplasm, not buffer+trap, so the load on the
motor is viscoelastic drag. The viscous part of this load differs by a
factor of 10,000-100,000 from the viscous load in an optical trap,
since the viscosity of water is .001 Pa*s and the zero-frequency
viscosity of cytoplasm is roughly 50 Pa*s. Does kinesin develop the
same force in these two environments? Our goal is to measure the
forces and work required to move vesicles in live cells and to
compare these to the limits established for kinesin in solution.
In the most basic T7 RNA polymerase (RNAP) experiment, a single fluorophore, covalently attached to GTP, will be incorporated at the 5' end of the RNA, thus marking the beginning of transcription (see figure). The signal from this incorporated fluorophore will persist until transcription terminates and the transcript diffuses away. By fitting histograms of the fluorescent persistence times, to kinetic models we can uncover essential information (average rate, processivity, abortive transcription percentage, etc.) regarding actively transcribing T7 RNAP.
One of the simplest known gene regulation systems is the autoregulation of lysozyme in T7 bacteriophage. T7 lysozyme is produced during a T7 infection to help lyse the bacterial cell wall in order to release the newly formed bacteriophage capsids. Additionally, T7 lysozyme autoregulates by inhibiting transcription by T7 RNAP. Using single-molecule fluorescence, we will observe the processive transcription rate of single T7 RNAP's in the presence of T7 lysozyme. Putative sequence dependence of the autoregulatory effect will also be examined.
Single molecule FRET is a powerful tool to probe mechanical motion in protein machines. In the FRET experiment (see figure), donor-labeled GTP will associate with the template DNA at the first position of transcription. Meanwhile, a his-tag, genetically engineered at the N-terminus of recombinantly expressed T7 RNAP, will bind tightly to the acceptor fluorophore at the N-terminal end of each surface-immobilized polymerase. In this way fluorescent transfer between donor and acceptor will begin immediately upon transcription initiation. The fluorescent signal from the donor and the energy transfer to the acceptor will suddenly drop if RNA transcription is aborted. Since aborts are quite common, many of the data traces will last only a fraction of a second. These short traces will be useful in characterizing the transition from initiation to elongation. For example, in a single molecule study of the E. coli Rep helicase, researchers found that the FRET signal oscillated markedly when the helicase paused on the DNA. Accordingly, we will look for a characteristic FRET signal in the abortive data that will shed light on the failed transition to elongation. Longer traces will also be informative: their FRET signal intensity should abruptly decrease at the transition between initiation and elongation. We will correlate this abrupt change to the initiation and the elongation crystal structures and help clarify the mechanical motions of this important transition.