may indicate p-cation interactions using two or more Tyr residues with the PC headgroup. 1863-Plat Lipid Bilayer Influences Rhodopsin Activation Probed by FTIR and UV-Visible Spectroscopy Blake Mertz 1 , Eglof Ritter 2 , Franz Bartl 2 , Michael F. Brown 3 . 1 West Virginia University, Morgantown, WV, USA, 2 Charite ´ Universita ¨tsmedizin, Berlin, Germany, 3 University of Arizona, Tucson, AZ, USA. Rhodopsin, the mammalian dim light photoreceptor, is the model protein for studying G protein-coupled receptor (GPCR) structure and function. We postulate that this process can be attributed to an ensemble of activated states - the ensemble activation mechanism (EAM) - and that the EAM is directly affected by membrane protein-lipid bilayer interactions dependent upon cell membrane composition, as predicted by the flexible surface model [1]. Rhodopsin was detergent-solubilized and incorporated into homogenous lipid vesicles (either POPC or DOPC) by dialysis. Samples were prepared at a series of temperature and pH values, bleached, and analyzed by simulta- neous Fourier transform infrared (FTIR) and UV-visible spectral acquisition. Activation in mixed-chain POPC bilayers drastically backshifts rhodopsin from the active Meta II photointermediate to the inactive Meta I state. A di-monounsaturated phospholipid like DOPC restores partial activity to rhodopsin, as well as stabilizing the protein in the Meta IIa state. Spectral reduction and analysis via pH titration curves yielded non-Henderson- Hasselbach behavior, indicating more than one activated state exists, thus supporting the concept of an EAM [2]. In addition, temperature changes have a marked effect on rhodopsin activation, decoupling the two protonation switches necessary for full activation at physiological temperatures. By manipulating the lipid environment, we validate the flexible surface model and EAM. Lipids with negative monolayer curvature such as DOPC facilitate rhodopsin activation towards the Meta II state [3]. Thermodynamic parame- ters showed that free energy changes are related to greater flexibility of rhodopsin in the cell membrane upon activation. Further validation of the flexible surface model is an important contribution to biophysical understand- ing of GPCR function. [1] A.V. Botelho et al. (2006) BJ 91, 4464-4477. [2] M. Mahalingam et al. (2008) PNAS 105, 17795-17800. [3] E. Zaitseva et al. (2010) JACS 132, 4815-4821. 1864-Plat Membrane Curvature Elastic Stress Strongly Modulates Metarhodopsin II Formation Olivier Soubias, Walter E. Teague, Jeff R. Dwulit-Smith, Kirk G. Hines, Klaus Gawrisch. NIH, Rockville, MD, USA. We studied the influence of curvature elastic stress in lipid monolayers in- duced by hydrophobic mismatch between lipid bilayer thickness and the hy- drophobic length of rhodopsin transmembrane helices on the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. Experiments were conducted at the low rhodopsin/lipid ratio of 1/1,000 to suppress rhodopsin oligomerization. Elastic stress was generated by reconstituting dark-adapted rhodopsin into a series of phosphatidylcholine (PC) bilayers with acyl chains of 14-20 carbons in length and cholesterol content varied from 0 to 30 mol %. 2 H NMR was used to monitor the adjustment of the length of hydrocarbon chains to the protein and to characterize monolayer curvature at the protein-lipid in- terface. We found that the average length of hydrophobic regions on rhodop- sin transmembrane helices is 2.6 5 0.1 nm. Thinner bilayers stretch and bend with negative monolayer curvature to match the length of hydrophobic heli- ces, while thicker bilayers get thinner with positive monolayer curvature near the protein. Shifts in the MI/MII equilibrium depended on the sign of monolayer curvature: negative curvature favored MI while positive curvature favored MII. The results suggest that MII formation generates negative curva- ture in monolayers near the protein, thus raising curvature stress for thin bilayers but releasing stress for thicker bilayers. Addition of cholesterol in- creases bilayer hydrophobic thickness, but otherwise showed identical behav- ior: bilayers with a hydrophobic thickness less than 2.6 nm favored MI while thicker bilayers favored MII. In case of severe hydrophobic mismatch between rhodopsin and bilayers, the behavior was complicated by rhodopsin oligomerization that seems to favor MI. The sensitivity of the MI/MII equilib- rium to negative curvature elastic stress was observed earlier in experiments with phosphatidylethanolamines by us and the Brown laboratory. Negative curvature stress upon MII formation is critical for understanding shifts in the MI/MII equilibrium. Platform: Transcription 1865-Plat Backtracking and Other Off-Path Pauses Control the Dynamic of Viral RNA Dependent RNA Polymerases David Dulin 1 , Igor D. Vilfan 2 , Bojk A. Berghuis 1 , Susanne Hage 1 , Dennis H. Bamford 3 , Minna Poranen 3 , Martin Depken 1 , Nynke H. Dekker 1 . 1 TU Delft, Delft, Netherlands, 2 Pacific Biosciences, Menlo Park, CA, USA, 3 University of Helsinki, Helsinki, Finland. RNA-dependent RNA Polymerases (RdRPs) are key players in the transcrip- tion and replication of RNA viruses. We study P2 of the bacteriophage F6 as a model system for structurally similar polymerases of e.g. positive ssRNA viruses such as Hepatitis C. During the different phases of the viral life cycle, P2 RdRP alternately transcribes or replicates viral RNA. Transcription pro- duces infectious positive-strand ssRNA whilst replication is the last step of the virus maturation in which the dsRNA genome is restored. Benefiting from the parallelism afforded by magnetic tweezers, we simulta- neously measure the transcription activity on tens of tethers while maintaining a resolution of 6 bases. In this way, we report on polymerase dynamics based on the analysis of an unusually large dataset (800 traces) taken under differing conditions of force and nucleotides concentration. This enables us to charac- terize P2 transcription elongation dynamics with unprecedented precision. Fits of this data to a kinetic model for polymerase activity via Maximum Likelihood estimation (MLE) reveal that the translocation step is insensitive to force and points to the existence of several types of off-pathway pause states in which the polymerase may be trapped. The associated pauses from short-lived exponentially-distributed pauses to long-lived pauses that follow a power law distribution over three decades and likely result from backtracking. By comparing our single-molecule data to the results of previous structural and biochemical studies on related RdRPs, we propose that the exponentially- distributed pauses are connected to the nucleotide selection process. Backtrack- ing has not been previously observed for the P2 RdRP, and our experiments show how it can be problematic for an RdRP, e.g. preventing it from complet- ing transcription or replication. 1866-Plat A Quantitative Kinetic Model of Eukaryotic Transcription Elongation from Single-Molecule Experiments Manchuta Dangkulwanich 1 , Toyotaka Ishibashi 1 , Shixin Liu 1 , Maria L. Kireeva 2 , Lucyna Lubkowska 2 , Mikhail Kashlev 2 , Carlos Bustamante 1 . 1 UC Berkeley, Berkeley, CA, USA, 2 National Cancer Institute, Fredrick, MD, USA. Transcription by RNA polymerase II (Pol II) is an important point of control for eukaryotic gene expression, and has been extensively studied by structural, biochemical, and biophysical methods. During transcription elongation, Pol II moves processively along the DNA template and synthesizes RNA, one nucle- otide at a time. A comprehensive kinetic characterization of this process, the transcription elongation cycle, incorporating both the on-pathway nucleotide addition phase and the off-pathway pausing phase, is still lacking. We used an optical trapping assay to follow transcription by individual yeast Pol II on bare or nucleosomal DNA. The single-molecule technique employed here allowed us to separate analyze the nucleotide addition phase and pausing phase separately, and arrived at a kinetic model that quantitatively describes both phases of transcription elongation. This model was used to measure the effects of a point mutation in the trigger loop motif on the on- and off- pathway dynamics of transcription, and can serve as a general framework to study the roles of various transcription and chromatin remodeling factors in transcription. 1867-Plat Computational Studies on Physical Mechanisms of T7 RNA Polymerase Elongation and Nucleotide Selection Jin Yu. Beijing Computational Science Research Center, Beijing, China. The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we study T7 RNAP as a model system for transcription elonga- tion. Based on structural information and experimental data from single- molecule force measurements, we had shown that a small translocation free energy bias aids initial nucleotide selection during elongation [1]. The selection is conducted by a conserved residue Tyr639 next to the active site. At the 364a Tuesday, February 5, 2013