Transcription constitutes the central point of control for gene expression in the cell. The eukaryotic Pol II enzyme must transcribe in the presence of the physical barriers present in nucleosomal DNA and both this enzyme and its prokaryotic counterpart must transcribe torsionally constrained DNA segments that require the enzymes to translocate against increasing torques. Moreover, during transcription elongation these two highly structurally homologous enzymes perform a mechanochemical cycle in which a nucleotide condensation reaction is coupled to the mechanical tasks of movement, force, and torque generation. In this application we propose to use a combination of imaging and single molecule manipulation approaches to: a) Investigate the dynamics of transcription through nucleosomal DNA by yeast Pol II. b) Determine the effect of elongation factors on nucleosomal transcription. c) Investigate the effect of histone modification on the dynamics of nucleosomal transcription. d) Investigate the effect of remodeling factors on the dynamics of Pol II through nucleosomal DNA. We also propose to continue our previous studies of the prokaryotic E. coli RNA polmerase enzyme to investigate: a) Its generation of torque during transcription of torsionally constrained DNA, and b) The details of its mechanochemical cycle at high spatial resolution.
Transcription constitutes the central point of control for regulation of gene expression. Disruption of this process is associated with congenital heart disease, oncogenesis, and a large variety of developmental pathologies. The insights that our experiments provide on transcription will support the first steps in the development of future therapies.
|Rollins, Geoffrey C; Shin, Jae Yen; Bustamante, Carlos et al. (2015) Stochastic approach to the molecular counting problem in superresolution microscopy. Proc Natl Acad Sci U S A 112:E110-8|
|Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos (2015) Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase. Elife 4:|
|Yen Shin, Jae; Lopez-Garrido, Javier; Lee, Sang-Hyuk et al. (2015) Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum. Elife 4:e06474|
|Mejia, Yara X; Nudler, Evgeny; Bustamante, Carlos (2015) Trigger loop folding determines transcription rate of Escherichia coli's RNA polymerase. Proc Natl Acad Sci U S A 112:743-8|
|Goldman, Daniel H; Kaiser, Christian M; Milin, Anthony et al. (2015) Ribosome. Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo. Science 348:457-60|
|Doniselli, Nicola; Rodriguez-Aliaga, Piere; Amidani, Davide et al. (2015) New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex. Nucleic Acids Res 43:5249-62|
|Kim, Hee-Kyung; Liu, Fei; Fei, Jingyi et al. (2014) A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation. Proc Natl Acad Sci U S A 111:5538-43|
|Ishibashi, Toyotaka; Dangkulwanich, Manchuta; Coello, Yves et al. (2014) Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics. Proc Natl Acad Sci U S A 111:3419-24|
|PressÃ©, Steve; Peterson, Jack; Lee, Julian et al. (2014) Single molecule conformational memory extraction: p5ab RNA hairpin. J Phys Chem B 118:6597-603|
|Llorente-Garcia, Isabel; Lenn, Tchern; Erhardt, Heiko et al. (2014) Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation. Biochim Biophys Acta 1837:811-24|
Showing the most recent 10 out of 102 publications