Investigation of RNA-Processing Protein Interactions and Dynamics with Simultaneous High-Resolution Tweezers and Fluorescence

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RNA is central to cellular information transfer, acting as the intermediate in the transformation of DNA-encoded information into protein. Essential for controlling this information flow is both production and degradation of RNA, processes that are carried out by energy-utilizing polymerase protein machines and exosomes, respectively. A third type of energy-utilizing RNA processing proteins are helicases, which are ubiquitous and modify the structure of RNA by unzipping RNA duplexes. An emerging theme is that RNA helicases are multi-functional machines playing additional roles beyond RNA unzipping, including energy requiring RNA anchoring and directly regulating the activity of other RNA processing proteins. The Ski2-like helicases are a new family of helicases, which act in close coordination with polymerases and the exosome to affect a broad range of RNA processing activities. These helicases have novel activity coupling RNA duplex sensing and motion. The project will investigate the fundamental molecular mechanisms of this novel activity to reveal how these diverse functions can arise from the accessory modification of core, conserved helicase components. The project will also investigate how these novel helicases cooperate and compete with other RNA processing proteins, e.g., a helicase and polymerase. The project will also leverage the optical tweezers developed and strong science outreach activities at MSU to expand an existing education and outreach program, which is based on hands-on interactions with biophysical science experiments at the levels of individual cells and RNA molecules both within and out of the lab. This project will investigate RNA-processing protein dynamics of single proteins and larger protein machine assemblies by developing and applying high-resolution, multi-modal single-molecule techniques. The project will specifically investigate the yeast Mtr4p helicase and the associated TRAMP helicase + polymerase system along with the related yet functionally distinct yeast DEAD-box helicase Ded1p. Mtr4p and TRAMP have emerged as key players in nuclear RNA surveillance and processing in part via precise modification of RNA for degradation by the exosome. While TRAMP is composed of only three proteins, two are opposing polarity molecular machines - the 3' to 5' Mtr4p helicase and the 5' to 3' Trf4p polymerase. Mtr4p performs multiple functions including unwinding RNA, discriminating substrates, and modulating polymerase activity to achieve precise poly(A) labeling of RNA. How these machines coordinate or compete to achieve function is controversial in large part due to their highly dynamic, multi-degree of freedom interactions on the scale of only 5-10 nucleotides of RNA. The project will be able to address the fundamental questions regarding how Mtr4p and TRAMP function using frontier single-molecule methods simultaneously combining high-resolution optical tweezers and fluorescence. The project will investigate: (1) How the recently discovered RNA-duplex-sensing translocation mechanism of Mtr4p is achieved via comparison to mutants, in particular one where the loss of a key domain dramatically alters activity, and to the related Ded1p helicase. (2) How the TRAMP polymerase activity is modulated in turn by Mtr4p via direct, simultaneous measurements of helicase and polymerase activity using fluorescence and tweezers. (3) How the related RNA helicase Ded1p unwinding mechanism occurs at the same mechanistic resolution as the Mtr4p studies. Advanced techniques combining high-resolution optical trapping and multi-color fluorescence will apply broadly across all biophysical molecular dynamics investigations towards the emerging need for tools to investigate protein-nucleic acid interactions beyond the individual molecule scale towards multi-protein, multi-degree of freedom complex systems. The project provides strong science outreach activities at MSU to expand an existing education and outreach program, which is based on hands-on interactions with biophysical science experiments at the levels of individual cells and RNA molecules both within and out of the lab.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.