Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

Abstract

The assembly of the E. 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.

Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment

“…For nucleic acids footprinting, dimethylsulfate was developed as a footprinting reagent for detection of protein-DNA interactions in cells (89,90); these approaches have been considerable improved over time (91,92). Radiolytic footprinting has recently been used to monitor ribosome assembly in bacteria through monitoring of RNA based protections as a function of assembly (93)(94)(95) providing novel and powerful readouts of complex cellular structures.…”

Toward Time-Resolved Analysis of RNA Metabolism in Archaea Using 4-Thiouracil

“…In this work, we provide proof of principle for the application of 4TU-based RNA-tagging in archaea. Given the known advantages of the methodology to analyze RNA dynamics and, RNA-protein interactions (e.g., Hafner et al, 2010 ; Ascano et al, 2012 ; Castello et al, 2012 ; Swiatkowska et al, 2012 ; Tallafuss et al, 2014 ; Duffy et al, 2015 ; Hulscher et al, 2016 ), we are confident that our proof of principle analysis broadens the archaeal molecular biology toolkit and will stimulate deeper analysis of RNA dynamics and gene expression networks in archaea.…”

“…Nucleotide analogs, such as 4-thiouracil- (4TU) or 4-thiouridine- (4SU), -based RNA-tagging has been successfully applied in various cell types and tissues in combination with high-throughput methodologies (e.g., Favre et al, 1986 ; Dölken et al, 2008 ; Zeiner et al, 2008 ; Friedel and Dolken, 2009 ; Miller et al, 2009 ; Tallafuss et al, 2014 ). Together, these studies have provided essential information for further understanding of RNA metabolism and its dynamics (e.g., Swiatkowska et al, 2012 ; Burger et al, 2013 ; Barrass et al, 2015 ; Duffy et al, 2015 ; Hulscher et al, 2016 ). Moreover, the recent improvement of RNA-tagging chemistry enables a better enrichment of the tagged-RNA population providing additional perspectives to more precisely characterize gene-expression networks ( Duffy et al, 2015 ).…”

SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline

“…The use of X-ray synchrotron-mediated hydroxyl radical footprinting (XFP) has increasingly gained interest in the past few years to probe the structure and dynamics of macromolecular complexes involving nucleic acids (Sclavi et al, 1997;Ralston et al, 2000) and proteins (Kiselar et al, 2002). XFP can also be used for in vivo analysis as recently reported for the study of ribosomal assembly (Clatterbuck Soper et al, 2013;Hulscher et al, 2016). With regard to proteins, synchrotron protein footprinting together with the development of high-resolution mass spectrometry (MS) for peptides emerges as a powerful structural proteomics technique that can shed light on the conformations and dynamics of macromolecular complexes in solution, and provides information at a single amino-acid residue resolution that was previously inaccessible using traditional approaches (Maleknia & Downard, 2014).…”