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. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.

Observation of intersubunit movement of the ribosome in solution using FRET.

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used Förster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.

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. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. We also discuss approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to specific 30 S protein residues have been used to determine distance changes within the 30 S subunit upon association with the 50 S subunit, forming the 70 S ribosome. Here, we explain the spectroscopic methods we have used, which should be of general interest in studies that aim at obtaining quantitative distance information from FRET.

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. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 S subunits and 70 S ribosomes from X-ray and cryo-electron microscope structures, and the platform is predicted to undergo a clock-wise rotation upon association.

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. The experiments and methods of data analysis are used in the lab section of a class on optical spectroscopy, where we go through the theory and applications of fluorescence. Because the fluorescence decay time of ruby is in the millisecond region, the instrumentation for this experiment can be constructed easily and inexpensively compared to the nanosecond-resolved instrumentation required for most fluorescent compounds, which have nanosecond fluorescence lifetimes. The methods are applicable to other luminescent compounds with decay constants from microseconds and longer, such as transition metal and lanthanide complexes and phosphorescent samples. The experiments, which clearly demonstrate the theory and methods of measuring temporally resolved fluorescence, are instructive and demonstrate what the students have learned in the lectures without the distraction of highly sophisticated instrumentation.

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.