Following the intersubunit conformation of the ribosome during translation in real time

Abstract

We report the direct observation of conformational rearrangements of the ribosome during multiple rounds of elongation. Using single-molecule fluorescence resonance energy transfer, we monitor the intersubunit conformation of the ribosome – in real time – as it proceeds from codon to codon. During each elongation cycle, the ribosome unlocks upon peptide bond formation, followed by reversion to the locked state upon translocation onto the next codon. Our data reveal both the specific and cumulative effects of antibiotics on individual steps of translation, and uncover the processivity of the ribosome as it elongates. Our approach interrogates the precise molecular events occurring at each codon of the mRNA within the full context of ongoing translation.

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

“…Both nonrotated and rotated state lifetimes are comparable with our previous reports (nonrotated lifetime = 5.5 ± 0.8 s and rotated lifetime = 3.9 ± 0.5 s in this study vs. nonrotated state lifetime = 2.6 ± 0.3 s and rotated-state lifetime = 2.0 ± 0.3 s on TIRF) (Fig. 4 C and D) (24). Decreased association rates are likely caused by steric and surface effects, but ribosomal function is clearly maintained (16).…”

“…3). The use of Cy3.5 and Cy5.5 instead of Cy2 used in prior experiments (16,24) gives signals that have greater SNR and longer lifetimes without excitation by an additional 488-nm laser. This improvement in dye photostability as well as the ability to use a wide range of fluorescent dyes allows robust single-molecule tracking of dynamics of multicomponent systems over a range of timescales from milliseconds to hours.…”

“…S6). Then, to show that the custom RS is capable of distinguishing subtle FRET transitions, we followed single Escherichia coli ribosomes as the two subunits (small 30S subunit and large 50S subunit) join during initiation and then rotate with respect to one another during translation, an assay that we have previously shown multiple times on TIRF (2,24). We immobilized 10 nM Cy3B-labeled 30S preinitiation complexes with aminoacylated initiator fMet tRNA (fMet-tRNA fMet ), initiation factor IF2, and a biotinylated mRNA [called 6(FK), which codes for an AUG start codon, a sequence of six alternating Phe and Lys codons, followed by a UAA stop codon], on the bottom of the ZMW.…”

“…4B). Each low-high-low intensity cycle (nonrotated to rotated and back to nonrotated state) corresponds to the ribosome translating one codon of the mRNA (24). Both nonrotated and rotated state lifetimes are comparable with our previous reports (nonrotated lifetime = 5.5 ± 0.8 s and rotated lifetime = 3.9 ± 0.5 s in this study vs. nonrotated state lifetime = 2.6 ± 0.3 s and rotated-state lifetime = 2.0 ± 0.3 s on TIRF) (Fig.…”

Where is the ‘Jennifer Aniston neuron’?

“…In order to experimentally test this hypothesis, the experiment requires controlling the electric field in addition to other characteristics and simultaneously record and analyze the protein response and protein-protein interactions. Given technological progress and innovations in wet labs (Blanchard, et al, 2004;Aitken and Puglisi, 2010) it has become possible to monitor in real time conformational dynamics and we expect any day soon to hear that the problem was completely solved. The propagation of action potential during a millisecond time, the charge density dynamics is not random, it is related to information processed and communicated by the neuron.…”