Ribosome RNA Assembly Intermediates Visualized in Living Cells

In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution SHAPE to probe the structure of E. coli 16S ribosomal RNA (rRNA) in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base paired structure different from that expected based on comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to pre-organize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes

Progress and challenges for chemical probing of RNA structure inside living cells

Proper gene expression is essential for the survival of every cell. Once thought to be a passive transporter of genetic information, RNA has recently emerged as a key player in nearly every pathway in the cell. A full description of its structure is critical to understanding RNA function. Decades of research have focused on utilizing chemical tools to interrogate the structures of RNAs, with recent focus shifting to performing experiments inside living cells. This Review will detail the design and utility of chemical reagents used in RNA structure probing. We also outline how these reagents have been used to gain a deeper understanding of RNA structure in vivo. We review the recent merger of chemical probing with deep sequencing. Finally, we outline some of the hurdles that remain in fully characterizing the structure of RNA inside living cells, and how chemical biology can uniquely tackle such challenges