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RNAP crystal structure with modeled DNA (Adapted from Korzheva et al., Science 2000)
A quick look under a microscope would convince anybody that despite the genetic identity of each of the human body's more than 200 cell types, there is a great morphological diversity between them. This diversity can be accounted for by differences in gene expression patterns in time and space. The central dogma of molecular biology describes the flow of genetic information from DNA (genes) to messenger RNA to enzymatically functional proteins. RNA Polymerase (RNAP) is the enzyme responsible for the first step in gene expression -- the DNA-directed synthesis of RNA -- known as transcription. During transcription RNAP, powered by the free energy released by nucleotide polymerization, can move along the DNA template at speeds greater than 10 nucleotides per second and can support forces greater than ~20pN (Yin et al. Science 1995). This capacity to sustain large forces explains RNAP's ability to displace bound proteins, such as histones, that it encounters as it transcribes along the DNA template. The ability to convert chemical energy into motion is similar to that of other motor proteins such as myosin and kinesin. However, the fact that RNAP moves along DNA -- a heteropolymer, as opposed to homopolymers such as actin or microtubules -- allows it to not only be regulated by extrinsic protein factors (lac, sigma, GreA/B, rho, etc.), but also by signals encoded within the DNA template (Ia- an arrest site in human histone genes) or RNA (trp terminator sites). This extra degree of regulation makes RNAP one of the most exquisitely controlled proteins in the cell. The regulation of gene expression can only be understood once we understand the detailed mechanism by which RNAP catalyzes the addition of a nucleotide to a growing RNA strand and couples this to its translocation along the DNA template.
Single molecule RNAP experiment using optical tweezers. RNAP is attached to a micron-sized polystyrene bead. The bead is then held in an optical trap equipped with an interference-based detection system. Tension is applied to the DNA, via an attachment to either the coverslip or a second bead (not shown). Transcription of RNAP affects the position of the bead.
The study of the mechanisms underlying RNAP transcription by conventional biochemical experiments, which report only ensemble averages, are complicated by the rapid loss of synchronization in populations of transcribing RNAP complexes. To overcome this difficulty, the concentration of NTPs is often reduced below physiological levels. Using optical traps we are able to track single transcribing molecules of RNAP at physiological NTP concentrations, thereby directly measuring kinetics and providing accurate spatial and temporal information. The trace below shows RNAP position versus time along the rpoB gene template. Optical trapping allows us to probe low probability short-lived events, such as the pauses in this trace, which would never be seen by traditional biochemical methods.
Single E. coli RNA polymerase molecule transcribing under force. The position of the polymerase in base pairs (bp) is shown as a function of time. The motion of the polymerase is frequently interrupted by pauses of variable duration, between which the transcriptional velocity is constant.
The optical trap also allows us to apply force to the transcribing RNA Polymerase. Provided that the force is not so great as to disturb the molecule, it can serve to tilt the energy landscape, perturbing the rates of force-dependent translocation steps. Because these rates in the biochemical cycle of nucleotide addition vary exponentially with applied force, the force versus velocity curve can teach us about the mechanism of translocation. The flatness of the force velocity curve below the stall force demonstrates that the rate limiting biochemical transition at zero-load is not force-dependent. The precipitous drop in the enzymatic velocity at higher forces can be explained by a large (5-10 base pair) transition, probably into an off-pathway state (Wang et al. Science 1997).
Averaged force-velocity relationships for RNAP. Force-velocity curve, ensemble average for 13 complexes in 1 mM NTP, 1 µM PPi. Individual force-velocity curves were normalized by their unloaded velocity and the force at which they reached half their unloaded velocity (Figure from Wang et al. Science 1997).
Recently, we've developed a two-bead assay for studying RNAP in which the RNAP is bound to one bead as above, but the end of the DNA is bound to another optically trapped bead (forming a "dumbbell"). Of course we don't see the RNAP or the DNA in the microscope, as seen below. If you're lucky you can catch us doing experiments LIVE on the Block Lab Webcam!
The RNAP Dumbbell experimental geometry. During normal elongation, RNAP (green) moves on the DNA (blue) as it elongates the nascent RNA (red). In the microscope we only see the two beads.
As the RNAP transcribes along the DNA, the two beads get closer together. Here is a movie of a single RNAP transcribing along the DNA at 1 mM ATP,CTP,GTP,UTP sped up 30 times [RNAP Dumbell 30x.avi 178 KB]. (During an experiment the position of the trap holding the left bead is adjusted to maintain a constant force on the right bead -- as if the RNAP was realing in the DNA, pulling the left bead towards it.)
RNAP transcription with the Dumbbell geometry. The position of the RNAP along the template during the movie is shown.