Large Effects of Discriminator Exchanges on the RNA Polymerase-Promoter Open Complex Structure, Lifetime and Transcription Initiation Patterns

Monday, February 29, 2016 transcription (Zygotic Genome Activation - ZGA) as a naturally occurring example of transcription level increase. General transcription activity occurs only following ZGA, i.e. 10 synchronous cell divisions after initial fertilization (three hours post fertilization). To make subnuclear distribution changes during ZGA accessible to super-resolution microscopy, we developed a dissociated cell culture protocol. Zebrafish embryos were dissociated at the 128-cell stage (three cell divisions before ZGA). Cultured cells developed transcription activity and morphological hallmarks of ZGA, confirming occurrence of native ZGA. We obtained preliminary results by wide-field fluorescence microscopy. Using fluorescently labeled histone proteins, which spontaneously bind DNA, we visualized a fine-grained chromatin structure in post-ZGA interphase nuclei in vivo. Utilizing antibody fragments (Fabs), we detected agglomerations of transcribing polymerase II in vivo. These agglomerations appeared as micron-sized, approximately spherical ‘‘transcription hot spots’’. Few large transcription hot spots occurred at the 256-cell stage; in consecutive cell cycles more and smaller spots appeared. Any distinct signal of transcribing polymerase II disappeared during cell divisions, when no transcription occurs, thus confirming specificity of Fab-based detection of actively transcribing polymerase II. In the future, we will employ super-resolution microscopy to resolve the fine structure of chromatin and transcription hot spots in vivo throughout native zebrafish ZGA. [1] Gavrilov and Razin, Molecular Biology, 2015. [2] Popken et al., Nucleus, 2015. 1156-Pos Board B133 Kinetics and Mechanism of Formation and Stabilization of the RNA Polymerase-Promoter Open Compex Munish Chhabra1, Raashi Sreenivasan1, Mikaela Poulos2, Emily Ruff3, Irina Artsimovitch4, Tom Record5. 1 Biophysics, University Of Wisconsin Madison, Madison, WI, USA, 2 University Of Wisconsin Madison, Madison, WI, USA, 3Chemistry, University Of Wisconsin Madison, Madison, WI, USA, 4Microbiology, Ohio State University, Columbus, OH, USA, 5Biochemistry, Biophysics and Chemistry, University Of Wisconsin Madison, Madison, WI, USA. Specific binding of E. coli RNA polymerase holoenzyme (RNAP; a2bb’s70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the
10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Fo¨rster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061). 1157-Pos Board B134 Large Effects of Discriminator Exchanges on the RNA PolymerasePromoter Open Complex Structure, Lifetime and Transcription Initiation Patterns Kate Henderson, Lindsey Felth, Si Wang, Cristen Molzahn, Munish Chhabra, Mikaela Poulos, Emily Ruff, Lauren Bieter, M. Thomas Record, Jr. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. Escherichia coli RNA polymerase holoenzyme (RNAP; consisting of a2bb’u þ s70 subunits) is the molecular machine of transcription. This machinery is set in motion by initial recognition of
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10 regions of linear promoter DNA by s70 and of the UP element region by the two flexiblytethered a-CTDs. A series of large conformational changes in both RNAP and DNA, driven by binding free energy, bend the downstream duplex into the active site cleft and open 13 base pairs including the
10 and discriminator regions and the transcription start site (þ1). Subsequently at some promoters the initial unstable (lifetime 1 s) open complex is stabilized by a network of interactions involving the discriminator DNA, s70 region 1.1, and downstream mobile elements (DME) of RNAP. These increase open complex lifetime to 6 minutes for T7A1 promoter and 17 hrs for the lPR promoter. An important

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but unanswered question is how the stability of the open complex affects its ability to initiate RNA synthesis upon binding of NTPs. Here we address this question by comparing lifetimes, structural features, and patterns of short and long RNA synthesized from lPR promoter with either the lPR or T7A1 discriminator, using nitrocellulose filter binding assays, permanganate footprinting and transcription assays. We also compare lifetimes and structural features of lPR , T7A1 and ribosomal rrnBP1 promoters with either lPR or T7A1 discriminator, as well as examine abortive and productive transcription products by acrylamide gel and novel mass spectrometric analysis to determine the extent to which the discriminator region is responsible for open complex lifetime, and to elucidate its effects on transcription initiation. This work is supported through funding by the NIH (GM103061). 1158-Pos Board B135 Effect of Pressure and Temperature on Transcription Initiation in Bacterial Cells Khanh Nguyen. University of Arkansas, Fayetteville, AR, USA. Abstract: In order for a cell to function normally in any environmental condition, a cell must be able to perform basic cellular functions such as cell metabolism, transcription, translation, and cell division. Adaptation to extremes of physicochemical conditions such as high pressure and temperature requires that all basic cellular processes are unrestricted. Here, we focus on the effect of pressure and temperature on transcription. For the initiation of transcription to occur, RNA polymerase must bind to DNA. We have studied the binding propensity of T7-RNA polymerase and Escherichia coli (E. coli) RNA polymerase to a consensus T7 promoter and E.coli recA promoter DNA, respectively. Specifically, we have investigated the stability of RNA polymerase-promoter DNA elongation complex in a wide range of pressure (1-2000 atm) and temperature (10-50oC) using fluorescence anisotropy. At constant temperature, the stability of elongation complex decreases with increasing pressure. The pressure at which transcription elongation stops depends on temperature. We construct a pressuretemperature phase diagram of the stability of elongation complex for both T7 polymerase þ T7 promoter and E.coli polymerase þ recA promoter. Furthermore, we show that the pressure-temperature stability phase diagram of elongation complex exhibits typical elliptical shape usually associated with the stability of proteins. Our results suggest that the evolution of polymerases has maintained their functionality in a wide range of pressures and temperatures. 1159-Pos Board B136 Measuring the Dynamics of TFIIF on RNA Polymerase II by smFRET Wei-hau Chang. Chemistry, Academia Sinica, Taipei, Taiwan. The making of mRNA transcripts in eukaryotes depends on an enzyme called RNA polymerase II. This enzyme is incapable of recognizing the promoter sequence on the DNA template until it forms a pre-initiation complex with a numerous transcription factors. Among the transcription factors, TFIIF binds tightly to RNA polymerase II and a recent X-ray structure of this complex shows it can stabilize the DNA template at the downstream and upstream regions. However, the density of TFIIF is missing in the X-crystal structure despite the DNA template is stabilized and suggests TFIIF is dynamical even when it is bound on RNA polymerase II in contrast to a static picture painted by a previous crosslink study. Here, we set out to quantify the movement of TFIIF relative to RNA polymerase II or the DNA template to better understand the function of TFIIF. In order to lengthen the observation window of the dynamics at single-molecule level, we introduce a donor carried by an off-resistant calmodulin on RNA polymerase II to create a FRET pair with an acceptor on various single-cysteine sites in TFIIF. 1160-Pos Board B137 Effect of FIS on Transcription-Coupled DNA Supercoiling in E. coli Samantha Dages, Xiaoduo Zhi, Fenfei Leng. Biochemistry, Florida International University, Miami, FL, USA. For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a ‘‘twin-supercoiled-domain’’ model of transcription in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. In this study, we utilized bacterial genetics and biochemical approaches to investigate how FIS protein regulates transcription-coupled DNA supercoiling (TCDS) by the strong rrnB P1, P2 promoters. We first generated several E. coli strains including MG1655(DE3)DfisDlacZ and VS111(DE3)DfisDlacZ using the l Red recombination system. We then inserted the divergently coupled Pleu-500 and