Aligning pitch for measurements of the shape of captive bubbles.

The first step required for the determination of surface tension from the shape of a captive bubble is the correct alignment of both the solid support against which the bubble floats and the camera used to record its profile. The solid support should be perpendicular to the gravitationally vertical axis. The camera used to visualize the bubble must be aligned to its axis of symmetry.

Optical factors in the rapid analysis of captive bubbles.

Bubbles and droplets offer multiple advantages over Langmuir troughs for compressing interfacial films. Experiments, however, that manipulate films to maintain constant surface tension (γ) present problems because they require feedback. Measurements of bubbles and droplets calculate γ from the shape of the interface, and calculations in real time based on finding the Laplacian shape that best fits the interface can be difficult.

Fitting experimental transcription data with a comprehensive template-dependent modular kinetic model.

In the companion article, we developed a modular scheme for representing the kinetics of transcription elongation by RNA polymerase. As an example of how to use these approaches, in this article we use a comprehensive modular model of this sort to fit experimental transcript elongation results obtained on the canonical tR2 template of phage λ by means of complementary bulk gel electrophoresis and surface plasmon resonance assays. The gel electrophoresis results, obtained in experiments quenched at various times after initiation of transcription, provide distributions of RNA lengths as a function of time.

Development of a "modular" scheme to describe the kinetics of transcript elongation by RNA polymerase.

Transcript elongation by RNA polymerase involves the sequential appearance of several alternative and off-pathway states of the transcript elongation complex (TEC), and this complicates modeling of the kinetics of the transcription elongation process. Based on solutions of the chemical master equation for such transcription systems as a function of time, we here develop a modular scheme for simulating such kinetic transcription data. This scheme deals explicitly with the problem of TEC desynchronization as transcript synthesis proceeds, and develops kinetic modules to permit the various alternative states of the TECs (paused states, backtracked states, arrested states, and terminated states) to be introduced one-by-one as needed.

The antitermination activity of bacteriophage lambda N protein is controlled by the kinetics of an RNA-looping-facilitated interaction with the transcription complex.

Protein N of bacteriophage lambda activates the lytic phase of phage development in infected Escherichia coli cells by suppressing the activity of transcriptional terminators that prevent the synthesis of essential phage proteins. N binds tightly to the boxB RNA hairpin located near the 5' end of the nascent pL and pR transcripts and induces an antitermination response in the RNA polymerase (RNAP) of elongation complexes located at terminators far downstream. Here we test an RNA looping model for this N-dependent "action at a distance" by cleaving the nascent transcript between boxB and RNAP during transcript elongation.

Monitoring RNA transcription in real time by using surface plasmon resonance.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed.

DNA multiplex hybridization on microarrays and thermodynamic stability in solution: a direct comparison.

Hybridization intensities of 30 distinct short duplex DNAs measured on spotted microarrays, were directly compared with thermodynamic stabilities measured in solution. DNA sequences were designed to promote formation of perfect match, or hybrid duplexes containing tandem mismatches. Thermodynamic parameters DeltaH degrees , DeltaS degrees and DeltaG degrees of melting transitions in solution were evaluated directly using differential scanning calorimetry.

Energetics and kinetics of cooperative cofilin-actin filament interactions.

We have evaluated the thermodynamic parameters associated with cooperative cofilin binding to actin filaments, accounting for contributions of ion-linked equilibria, and determined the kinetic basis of cooperative cofilin binding. Ions weaken non-contiguous (isolated, non-cooperative) cofilin binding to an actin filament without affecting cooperative filament interactions. Non-contiguous cofilin binding is coupled to the dissociation of approximately 1.