In vivo biochemistry in bacterial cells using FRAP: insight into the translation cycle.

In vivo measurements of the mobility and binding kinetics of cellular components are essential to fully understand the biochemical processes occurring inside cells. Here, we describe a fluorescence recovery after photobleaching-based method that can be easily implemented to the study of reaction-diffusion processes in live bacteria despite their small size. We apply this method to provide new, to our knowledge, quantitative insight into multiple aspects of the bacterial translation cycle by measuring the binding kinetics and the micrometer-scale diffusive properties of the 50S ribosomal subunit in live Caulobacter cells. From our measurements, we infer that 70% of 50S subunits are engaged in translation and display, on average, limited motion on the micrometer scale, consistent with little mixing of transcripts undergoing translation. We also extract the average rate constants for the binding of 50S subunits to 30S initiation complexes during initiation and for their release from mRNAs when translation is completed. From this, we estimate the average time of protein synthesis and the average search time of 50S subunits before they engage in the next initiation event. Additionally, our experiments suggest that so-called free 50S subunits do not diffuse freely; instead their mobility is significantly slowed down, possibly through transient associations with mRNA.