RNA Polymerase as a Molecular Motor

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The existing single-molecule studies of E. coli RNAP suggest a number of promising avenues for future research into transcription mechanisms in both prokaryotes and eukaryotes. First, if measurements can be made precisely enough to resolve 1 bp steps with high time resolution, the experiments could discriminate between alternative translocation mechanisms and reveal essential features of RNAP chemomechanical coupling. In particular, such studies could differentiate between Brownian ratchet and power stroke translocation mechanisms since these models make quantitatively different predictions about the way that translocation step durations vary with applied force. Visualizing RNAP movements with single–base pair precision would also settle the question of whether >1 bp sliding movements are characteristic features of the ordinary chain elongation cycle. Second, single-molecule approaches are ideally suited to examining the differences in reaction kinetics between TECs in the same population that are following different, parallel reaction pathways (Erie et al. 1993xErie, D.A, Hajiseyedjavadi, O, Young, M.C, and von Hippel, P.H. Science. 1993; 262: 867–873CrossRef | PubMedSee all ReferencesErie et al. 1993) or that have heterogeneous structures. Third, single-molecule methods can not only detect movement of RNAP along the DNA, but also directly visualize RNAP attachment to and release from DNA. Therefore, the techniques can visualize transcription initiation and termination, making them powerful tools to investigate mechanisms of transcription regulation and the control of gene expression. Fourth, these techniques can visualize the large-scale structural changes in DNA or chromatin associated with transcription or transcription regulation. These include DNA looping (e.g.,3xFinzi, L and Gelles, J. Science. 1995; 267: 378–380CrossRef | PubMedSee all References, 12xRippe, K, Guthold, M, von Hippel, P.H, and Bustamante, C. J. Mol. Biol. 1997; 270: 125–138CrossRef | PubMed | Scopus (122)See all References), DNA bending (e.g.,Rippe et al. 1997xRippe, K, Guthold, M, von Hippel, P.H, and Bustamante, C. J. Mol. Biol. 1997; 270: 125–138CrossRef | PubMed | Scopus (122)See all ReferencesRippe et al. 1997), and chromatin rearrangements (seeFritzsche et al. 1995xFritzsche, W, Vesenka, J, and Henderson, E. Scanning Microsc. 1995; 9: 729–737PubMedSee all ReferencesFritzsche et al. 1995references therein). Single-molecule techniques can potentially give a more detailed structural picture than biochemical (e.g., gel-shift) methodologies and can observe changes in structure with high (in some cases, millisecond) time resolution.Perhaps the most important future applications of single-molecule microscopy techniques are analyses of biochemical pathways that involve assembly of large macromolecular complexes. Transcription and transcription regulatory systems involve the assembly of complex structures consisting of multiple protein molecules that interact with each other and with sites on the DNA and RNA. Despite the importance of these systems, in few cases has it been possible to define fully the kinetic mechanism of assembly (that is, the complete pathway of assembly and the rates of all steps) and its temporal relationship to catalytic and regulatory events. Recently, technology has been developed to visualize by fluorescence microscopy single protein molecules tagged with small organic dyes or expressed as fusions with green fluorescent protein (Funatsu et al. 1995xFunatsu, T, Harada, Y, Tokunaga, M, Saito, K, and Yanagida, T. Nature. 1995; 374: 555–559CrossRef | PubMedSee all ReferencesFunatsu et al. 1995). This should permit observation of the assembly of multiple proteins (perhaps each tagged with a different color fluorophore) into single transcription or regulatory complexes while translocation is simultaneously observed using one of the techniques described above. The technology is thus particularly well-suited to ask questions about the relationship between elongation factors binding to transcription complexes and their effects on the rate and persistence of transcription, because both the factor binding and kinetic changes can be individually monitored. For example, such studies could answer fundamental questions about the mechanisms of antitermination in bacteria, and in eukaryotes could reveal the temporal relationships between transcription complex assembly, regulatory factor binding, promoter escape, and conversion to an elongation-proficient TEC. By using polarization optics, single-molecule fluorescence microscopy can also directly observe reorientation of single dye molecules relative to the excitation light (Sase et al. 1997xSase, I, Miyata, H, Ishiwata, S, and Kinosita, K Jr. Proc. Natl. Acad. Sci. USA. 1997; 94: 5646–5650CrossRef | PubMed | Scopus (138)See all ReferencesSase et al. 1997). It thus has the potential to detect not only binding events but also structural reorganizations within single macromolecular complexes.Analysis of RNAPs as molecular motors is still in its infancy. Single-molecule experiments should improve our knowledge of the fundamental mechanisms of transcription and its regulation, particularly as the techniques become more widely used and better instrumentation is developed. These methods should be of value to study other DNA-based motor enzymes, including DNA polymerases, exo- and endonucleases, helicases, and topoisomerases.