The antibiotic viomycin traps the ribosome in an intermediate state of translocation.

During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation.

Ruby crystal for demonstrating time- and frequency-domain methods of fluorescence lifetime measurements.

We present experiments that are convenient and educational for measuring fluorescence lifetimes with both time- and frequency-domain methods. The sample is ruby crystal, which has a lifetime of about 3.5 milliseconds, and is easy to use as a class-room demonstration.

Properties of microfluidic turbulent mixing revealed by fluorescence lifetime imaging.

We present a new method of measurement based on fluorescence lifetime imaging that reveals molecular-scale details of the mixing process in a continuous-flow turbulent microfluidic reactor. Our data provide a glimpse of the cascade to the minimal eddy size, followed by rapid diffusion involving the smallest eddies for final mixing.

Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer.

We have used Förster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution.

Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.

We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site.