| RNA molecules play essential roles in diverse cellular functions. Most of these functions depend on the ability of the RNA to fold into a stable structure. In the cell, endogenous RNAs likely adopt many intermediate structures before the final functional structure is formed. These structural changes occur in an environment very different from that of most in vitro explorations of RNA structure-function relationships. For example, the ribosome is a dynamic ribonucleoprotein responsible for synthesizing all the proteins in cells. Ribosomal RNA (rRNA) has been implicated in plying a crucial role in the structural, catalytic, and dynamic aspects of translation. Therefore, it is critical to be able to directly probe RNA structure in its native environment, the cell. RNA SHAPE technology provides accurate and quantitative RNA structure information and is ideal to being adapted to in vivo conditions. In this work I first investigate the mechanism of SHAPE so that we have a better understanding of this important technology. I then show that SHAPE technology can be used to probe RNA structure in vivo . I then use this new technology to obtain structural information for over 1400 16S nucleotides in many conformational states of the 16S rRNA in vivo. In many regions, the in vivo SHAPE chemistry is exactly consistent with in vitro RNA structure studies. However, in several key areas, the in vivo rRNA structure differs from that of its in vitro counterpart. Local, but highly significant, rearrangements in the RNA secondary structure thus appear to contribute to ribosome function in vivo. As further evidence that investigating RNA structure in vivo is necessary for our fundamental understanding of biology, I use in vivo SHAPE to identify a novel secondary mode of action for an antibiotic. This work illustrates that a full understanding of dynamic RNA structure at nucleotide resolution will be critical for complete understanding of RNA function in vivo. |
