Using Hydroxyl Radical Footprinting to Observe Ribosome Assembly Intermediates in vivo

Abstract

The assembly of the E. coli ribosome small subunit has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells remains poorly understood. This is a very complex process in which an rRNA is transcribed, folded, cleaved, and modified, while also binding with 20 different proteins. Very little is known about how the tertiary structure of the ribosomal RNA changes during assembly. There are a number of structure-probing methods that can be used to study rRNA in vivo, but virtually all of them lack the time resolution necessary to study a process like ribosome synthesis, which is completed within a few minutes.Hydroxyl radical footprinting can be used to probe in vivo rRNA structure. The hydroxyl radicals which probe the rRNA can be produced in milliseconds using synchrotron X-rays. With this technique it is possible to examine ribosome assembly with meaningful time resolution. The hydroxyl radicals cleave the RNA backbone in solvent accessible regions, giving cleavage patterns that reflect regions of flexibility and rigidity within an RNA.For the purpose of examining ribosome assembly, it is nascent ribosomes that are of interest, not pre-existing ribosomes that are already assembled. Therefore, the nascent ribosomes must be isolated from the background of pre-existing ribosomes. It has been shown that cells can take up labeled nucleosides that have been added to their growth media and incorporate them into nascent RNA transcripts. These can then be isolated using affinity methods. Once the nascent, assembling rRNA has been isolated, it can be analyzed by primer extension. The reverse transcriptase terminates at the cleavage sites. The cDNA fragments are then able to be analyzed by either slab gel, capillary electrophoresis, or high-throughput sequencing methods.