Structure-Function Principles of RNA in Cotranscriptional Folding Pathways
Luyi is a 2nd year IBiS student in Julius Lucks' lab. She received a BS in Biochemistry from University of Washington.
While RNA acts as a messenger between DNA and protein within the central dogma, the single-stranded nature of RNA leads way to unique secondary and tertiary structures responsible for catalytic or regulatory abilities in non-coding RNA. Many of these functionally-significant structures are achieved in nascent RNA being folded as it is actively being transcribed and emerging from the Polymerase. To study the structures of RNA during cotranscriptional folding, we turn to transcriptional riboswitches found in biology. Riboswitches are self-regulatory elements found in bacterial organisms that control gene expression in response to a specific ligand. They do this by “switching” between two possible RNA structures in a ligand-dependent fashion. The final structures typically consist of a terminated “off” structure that represses gene expression, or an anti-terminated “on” structure that will fully transcribe and activate gene expression. This behavior makes riboswitches a suitable model system where we can control the input, then observe how the structures in a resulting folding pathway can cause a functional output. The dynamic structures adopted by riboswitches during cotranscriptional folding have been shown to be under kinetic control and differ from thermodynamic minimum free-energy folded conformations, highlighting the need for a way to uncover the structures to study their significant role in dictating function.
To map intermediate RNA structures during cotranscriptional folding, I use selective 2’-hydroxyl acylation analyzed by primer extension and sequencing (SHAPE-Seq), a technique developed by the Lucks Lab. With SHAPE-Seq, RNAs of varying lengths are first transcribed, chemically probed, then reverse transcribed and sequenced. The fragment length distribution of cDNAs enables us to infer the reactivity, and therefore, the structure around individual nucleotides. Combined with next-generation sequencing, we can then acquire data of an entire population of RNA in a high throughput manner, allowing us to deduce the structures of RNAs transcribed at varying lengths to map the folding pathways of riboswitches at nucleotide-resolution levels. This mechanistic detail can help to elucidate the biophysical mechanisms for overarching structure-function relationships of RNA that are known to control fundamental cellular processes.