Previous studies have indicated that the nucleosome poses a barrier to the passage of RNA polymerase. It is now shown that a trailing bacterial RNA polymerase can reduce backtracking and enhance the movement of a leading polymerase through the nucleosomal barrier. During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10–base pair periodicity and backtracked by ∼10–15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAP's elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

The Endosomal Sorting Complexes Required for Transport III (ESCRT-III) proteins are critical for cellular membrane scission processes with topologies inverted relative to clathrin-mediated endocytosis. Some viruses appropriate ESCRT-IIIs for their release. By imaging single assembling viral-like particles of HIV-1, we observed that ESCRT-IIIs and the ATPase VPS4 arrive after most of the virion membrane is bent, linger for tens of seconds, and depart ~20 s before scission. These observations suggest that ESCRT-IIIs are recruited by a combination of membrane curvature and the late domains of the HIV-1 Gag protein. ESCRT-IIIs may pull the neck into a narrower form but must leave to allow scission. If scission does not occur within minutes of ESCRT departure, ESCRT-IIIs and VPS4 are recruited again. This mechanistic insight is likely relevant for other ESCRT-dependent scission processes including cell division, endosome tubulation, multivesicular body and nuclear envelope formation, and secretion of exosomes and ectosomes.

Particle interactions were directly determined from the lateral compression of two-dimensional plasma dust crystals confined in a parabolic potential well. Measured particle separations combined with an equation of state for the crystal were used to derive values for the particle screening length and the charge on the particles. The shape of the parabolic potential well was confirmed by observing trajectories of single particles falling within the well. No evidence of a lateral particle-particle binding was observed.

Total internal reflection fluorescence (TIRF) microscopy is a high‐contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z‐resolution of an epi‐fluorescence microscope. This technique also increases signal‐to‐noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through‐the‐objective TIRF illuminator and a prism‐based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through‐the‐objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi‐ and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed. Curr. Protoc. Cytom. 61:12.29.1‐12.29.19. © 2012 by John Wiley & Sons, Inc.