Abstract: The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.
Figure 3 from Hulscher et al: Apparatus for X-ray footprinting of live cultures (a) Schematic showing the flow of liquid culture past the X-ray beam and into a fraction collector. A syringe pumo is used to dispense nutrients into the culture at the desired time. A MSD pump displaces culture through a capilary flow cell in the path of the X-ray bean, at flow rates up to 5 mL/min. The X-ray dose depends on the flow rate, the length of tubing exposed to the beam (horizontal shift), and the X-ray flux density of the beam itself. The esposed culture is colelcted in 0.75 - 1 mL fractions. (b) Photograph of apparatus at NSLS X2BC. The X-Ray beam pope is pointint toward the fron of the image.
Results from: Hulscher RM, Bohon J, Rappé MC, Gupta S, D'Mello R, Sullivan M, Ralston CY, Chance MR, Woodson SA. Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting. Methods. 2016 Jul 1; 103:49-56. doi: 10.1016/j.ymeth.2016.03.012. Epub 2016 Mar 22. PMCID: PMC4921310.