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The three-dimensional structure of the translating 70S E. coli ribosome is presented in its two main conformations: the pretranslocational and the posttranslocational states. Using electron cryomicroscopy and angular reconstitution, structures at 20 A resolution were obtained, which, when compared with our earlier reconstruction of "empty" ribosomes, showed(More)
Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed in live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a three-dimensional FRAP model that accounts for a single spatially localized cluster of binding sites in the(More)
Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor occupancy, but give no indication of kinetics and are(More)
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables(More)
We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm(More)
Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)--especially in rapidly inducible systems--is largely unknown. Using fluorescence in situ hybridization and computational models, we(More)
Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient(More)
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z-scanning and is often too slow to capture biological events. We report a new aberration-corrected multi-focus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multi-focus system enables(More)