# The RNA nanomachines of gene expression dissected at the single molecule level > **NIH NIH R35** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $975,089 ## Abstract ABSTRACT: Since 2000, the PI has been awarded continuous NIH funding to contribute to the RNA field by building a broad research portfolio focused on dissecting the mechanisms of the nanoscale RNA machines of gene expression – ranging from small ribozymes and riboswitches to the RNA silencing machinery – by single molecule fluorescence microscopy. Building on this expertise, the two long-term goals of the current MIRA renewal proposal are to: 1.) Apply our established mechanistic enzymology approaches to an ever broader set of RNAs involved in regulating mRNA transcription and translation, while seizing opportunities arising from the continuing discoveries of new RNA functions. 2.) Push the technical limits of our approaches to be able to probe increasingly complex biological machineries and mechanisms since unexpected discoveries – as we found – often await where RNA nanomachines interact. In pursuit of these goals, we will address the overarching hypothesis that both secondary and tertiary structures of RNA determine the outcomes of gene expression, in often enigmatic and poorly understood ways that were traditionally overlooked by a field rooted in genetics, where genes to this day often are drawn as sequence strings without structural features. Such thinking is countered by, for example, the fact that nascent RNA structures have a significant impact on transcription in the form of regulatory riboswitches embedded near the 5' ends of bacterial mRNAs that respond to specific ligands by forming transcription terminator hairpins. In addition, the time-ordered, 5'-to-3' directional RNA synthesis during transcription often yields kinetically trapped RNA folds distinct from the most thermodynamically stable structure of a refolded full-length transcript. Encapsulating the power of our pursuit, we are combining our signature single-molecule, biochemical and computational approaches to show that bacterial transcription pausing frequently occurs at sites immediately downstream of both transcriptional and translational riboswitches, which allows riboswitch ligands to affect transcription and transcription-translation coupling, respectively. In addition, we are interrogating the mechanism of mammalian RNA silencing using single particle tracking both in cultured human cells and reconstituted in human cell extracts. We posit that our work will continue to discover more examples of intimate structural and kinetic coupling between RNA folding and gene expression, leading to the exquisite gene regulation underlying all life processes. Ultimately, we anticipate that our studies have the potential to transform our understanding of RNA structure-function relationships in general, and of how RNA secondary and tertiary structure is governing the function of cellular gene expression machines in particular. ## Key facts - **NIH application ID:** 10840519 - **Project number:** 2R35GM131922-06 - **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR - **Principal Investigator:** NILS G WALTER - **Activity code:** R35 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $975,089 - **Award type:** 2 - **Project period:** 2019-05-01 → 2029-04-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10840519 ## Citation > US National Institutes of Health, RePORTER application 10840519, The RNA nanomachines of gene expression dissected at the single molecule level (2R35GM131922-06). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10840519. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*