RNA polymerase slides home: pause and termination site recognition.

At least four caveats apply to the sliding-clamp model. First, just as Chamberlin 1995xSee all ReferencesChamberlin 1995 noted of the inchworm model, sliding has been detected only in halted TCs. At present, we cannot be certain how these movements relate to events that occur on the rapid timescale of transcription. It is formally possible that inchworm-like, flexible movements do occur as the TC elongates rapidly and lead to sliding once TCs are halted (a reversal of the current cause–effect interpretation). Further, although rapid fluctuations in the lateral position of the TC are an appealing explanation for changes in step-time and apparent Km for NTPs during elongation, whether oscillations are possible on the timescale of transcription is untested.Second, RNAP activity in vivo is modulated by elongation factors (e.g., NusA and NusG in E. coli), and we do not yet know their effects on the lateral stability of RNAP.Third, the sliding clamp model does not explain the remarkable ability of the TC to remain intact and active when a replication fork passes it (Liu et al. 1993xLiu, B., Wong, M.L., Tinker, R.L., Geiduschek, E.P., and Alberts, B.M. Nature. 1993; 366: 33–39Crossref | PubMedSee all ReferencesLiu et al. 1993). This has been cited as strong evidence for the inchworm model and the unimportance of an RNA:DNA hybrid. In theory, sliding of RNAP onto newly replicated DNA could allow a replication fork to pass if DNA synthesis restarts at a 3′ terminus as it emerges from a sliding RNAP (DNA polymerases are distributive; i.e., they can rebind and extend released 3′ ends). However, TCs containing 18 nt RNAs (which can presumably slide at most 10 bp) survive replication passage (Liu et al. 1993xLiu, B., Wong, M.L., Tinker, R.L., Geiduschek, E.P., and Alberts, B.M. Nature. 1993; 366: 33–39Crossref | PubMedSee all ReferencesLiu et al. 1993). At best, this could allow replication through a TC without disruption of an 8 bp hybrid, but still demands significant disruption of contacts within the ∼35 bp RNAP footprint on DNA.Fourth, some evidence exists for structural flexibility in RNAP. Stretching between the active site and another part of RNAP must be possible because an initiating nucleotide crosslinked to His-1237 of the β subunit can be extended by up to 8 nt (Mustaev et al. 1993xMustaev, A., Kashlev, M., Zaychikov, E., Grachev, M., and Goldfarb, A. J. Biol. Chem. 1993; 26: 19185–19187See all ReferencesMustaev et al. 1993). A comparable experiment on crosslinked RNA in an elongating TC has yet to be reported. The ability of RNAP to extend its RNA chain 7 nt closer to EcoRI or LacI roadblocks than their individual footprints predict is an additional suggestion of flexibility (Guerin et al. 1996xGuerin, M., Leng, M., and Rahmouni, A.R. EMBO J. 1996; 15: 5397–5407PubMedSee all ReferencesGuerin et al. 1996references therein).Important questions remain. If the sliding-clamp model is correct, what properties of RNAP's downstream DNA clamp allow it to slide freely along DNA but to interact specifically at pause and termination sites? What is the structure of the RNA:DNA hybrid? How far and how fast can RNAP spontaneously slide along the RNA and DNA chains? Does structural flexibility exist in RNAP and does it contribute to events during chain elongation? Do all TCs at a given template position have the same potential for sliding, or do different, perhaps slowly interconverting, populations exhibit different properties during chain elongation? (a key implication of the inchworm model; seeUptain et al. 1997xUptain, S., Kane, C., and Chamberlin, M. Annu. Rev. Biochem., in press. 1997; See all ReferencesUptain et al. 1997references therein).Answering these questions will require even more powerful experimental approaches. Immediate priorities should be (i) the use of rapid footprinting and crosslinking techniques or high-resolution single-molecule microscopy to examine changes in RNAP structure and DNA position on the timescale of normal chain elongation, (ii) elucidation of TC structure by electron or X-ray diffraction of crystals of RNAP, and (iii) study of RNAP movements in the presence of elongation factors and conditions closer to those in vivo.