NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 15 NUMBER 8 AUGUST 2008 779 molecular dynamics simulations of the yeast Pol II NAC suggest that the direction of the attack is reversed in such a way that it is the substrate bound to the mobile trigger loop that attacks the static nucleophile (RNA) 14 . Thus, the bridge helix and trigger loop can switch between ‘leading’ and ‘assisting’ roles during translocation and catalysis. An integrated NAC model can now be proposed (Fig. 2) that incorporates these views and indicates the steps where α-amanitin could interfere with RNA synthesis. In summary, the work of the Cramer and Kornberg laboratories reveals previously unobserved conformations and states of RNAP trapped by the transcriptional inhibitor α-amanitin. They provide a new understanding for the role of the catalytic center mobile elements in the NAC and pave the way for future investigations. ACKNOWLEDGMENTS This work was supported by grants from the US National Institutes of Health. 1. Borukhov, S. & Nudler, E. Trends Microbiol. 16, 126–134 (2008). 2. Kaplan, C.D., Larsson, K.M. & Kornberg, R.D. Mol. Cell 30, 547–556 (2008). 3. Brueckner, F. & Cramer, P. Nat. Struct. Mol. Biol. 15, 811–818 (2008). 4. Bushnell, D.A., Cramer, P. & Kornberg, R.D. Proc. Natl. Acad. Sci. USA 99, 1218–1222 (2002). 5. Bar-Nahum, G. et al. Cell 120, 183–193 (2005). 6. Kireeva, M.L. et al. Mol. Cell 30, 557–566 (2008). 7. Toulokhonov, I., Zhang, J., Palangat, M. & Landick, R. Mol. Cell 27, 406–419 (2007). 8. Wang, D. et al. Cell 127, 941–954 (2007). 9. Vassylyev, D.G. et al. Nature 448, 163–168 (2007). 10. Zhu, R. et al. Theor. Chim. Acta 120, 479–489 (2008). 11. Bai, L., Fulbright, R.M. & Wang, M.D. Phys. Rev. Lett. 98, 068103 (2007). 12. Vassylyev, D.G. et al. Nature 417, 712–719 (2002). 13. Campbell, E.A. et al. EMBO J. 24, 674–682 (2005). 14. Zhu, R. & Salahub, D.R. AIP Conf. Proc. 963, 104–110 (2007). that mutations in the trigger loop affected bridge helix conformations as well as enzyme translocation, fidelity and response to regulatory signals and factors 5 . Bridge helix conformational changes in that work were observed directly using cross-linking approaches. However, the particular structural manifestations of the bridge helix–centric thermal ratchet (bending and straightening of the bridge helix) seemed to be a result of an earlier crystallographic aberration, which in turn called into question the significance of the bridge helix element in RNAP function (the extreme trigger loop– centric model discounted the importance of the bridge helix altogether 7 ). The work of Brueckner and Cramer restores the role of the bridge helix as crucial in RNAP translocation and provides a more accurate view of the structural changes (shifting of the central portion of bridge helix) that accompany the repositioning of the DNA template to a new, ‘pretemplating’ position in an intermediate translocation state. The movement of the bridge helix in and out of the pretemplating position may depend on the wedged trigger loop, which can push the bridge helix into position, stabilize the shifted bridge helix or act passively as a boundary condition directing the distortion of the bridge helix by a force arising elsewhere. Thus, bridge helix oscillation could still be a structural manifestation of the Brownian ratchet in translocation. Swinging of the refolded substrate-bound trigger loop, which takes the shape of an α-helical hairpin packed against the bridge helix, toward the insertion site could provide the initial velocity and directionality of the nucleophilic attack of the RNA 3′-OH on the α-phosphate of the substrate NTP. Indeed, reduced accuracy 2 ), or even an artifact caused by the particular scaffold design, absence of NTP or other factors. The importance of Met932 for bacterial transcription cannot be unequivocally associated with its role in wedging of the trigger loop into the bridge helix, because its counterpart Met1238 was also reported to stack on the substrate base in the T. thermophilus EC structure 9 , and its substitution may have unknown effects on trigger loop mobility and/or refolding. Similar substitution of a nearby Thr934 had an even more drastic effect (22-fold) on pausing without any direct involvement in the formation of the wedge 7 . Regardless of the extent to which this wedged conformation represents an in- pathway translocation intermediate, Brueckner and Cramer have made several important contributions to our understanding of how RNAP functions. The new structure of yeast Pol II EC allows for more rigorous molecular dynamics simulations of transcription elongation, which, together with structural studies, bulk and single-molecule biochemistry, and kinetic analysis, are needed to generate an explicit physical model of transcription. Direct observation of the equilibrium between the pre- and post-translocated ECs lends further support to the concept of RNAP as a Brownian ratchet. This work also attempts a synthesis of bridge helix–centric and trigger loop–centric models of elongation by emphasizing the concerted movements of the bridge helix and trigger loop during translocation. Broad acceptance of the Brownian ratchet mechanism of multisubunit RNAPs (as opposed to a power-stroke mechanism) began with the bridge helix–centric model of Bar-Nahum et al., who demonstrated bind to specific sequences in the target pre- mRNAs, known as exonic (ESE) or intronic (ISE) splicing enhancers depending on their location. Some SR proteins are concentrated in nuclear subcompartments called speckles, from where they migrate to transcription sites upon transcriptional activation 2 . The SR protein SC35 was discovered and characterized for its important role in constitutive and alternative splicing, and its Both constitutive and alternative splicing require SR proteins, not only for the recruitment of general splicing factors involved in the assembly of functional spliceosomes, but also for bridging factors sitting at 3′ and 5′ splice sites that define alternative exons 1 . SR proteins are functionally modular: a C-terminal domain rich in arginines and serines (RS domain) mediates interactions with other proteins, whereas one or two N-terminal RNA recognition motifs (RRMs) Serine/arginine-rich (SR) proteins are a conserved family of proteins primarily known for their numerous roles in pre-mRNA splicing. A splicing regulator promotes transcriptional elongation Juan Pablo Fededa & Alberto R Kornblihtt A new study reveals that the serine/arginine-rich splicing factor SC35 is necessary to promote RNA polymerase II elongation in a subset of genes, confirming a bidirectional coupling between transcription and splicing. Juan Pablo Fededa and Alberto R. Kornblihtt are at the Laboratorio de Fisiología y Biología Molecular, IFIByNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina. e-mail: ark@fbmc.fcen.uba.ar NEWS AND VIEWS © 2008 Nature Publishing Group http://www.nature.com/nsmb