Title

THE MTR4 RATCHET HELIX FUNCTIONS IN CONCERT WITH THE ARCH DOMAIN TO REGULATE HELICASE ACTIVITY

Document Type

Presentation

Publication Date

4-10-2014

Faculty Mentor

Sean Johnson

Abstract

Mtr4 is a conserved superfamily-2 RNA helicase that activates exosome mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. As a member of the Trf4-Air2-Mtr4 polyadenylation (TRAMP) complex, Mtr4 is known to unwind polyadenylated RNA substrates and present them to the exosome for degradation. Prominent features of the Mtr4 structure include a four domain ring like helicase core and a large arch domain that spans the core. Within the helicase core, a "ratchet helix" is positioned to interact with the bases of unwound RNA substrates. However, the contribution of these interactions to Mtr4 activity is poorly understood. Here we show that conservation along the ratchet helix is particularly extensive for Mtr4 as compared to related helicases. Mutation of residues along this helix alter RNA unwinding rates in both an Mtr4 and TRAMP context, and result in slow growth phenotypes in vivo. We further observe that R1030 on the ratchet helix influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain had minimal effect on Mtr4 unwinding activity. Surprisingly, we now show that the combination of archless and ratchet helix mutants completely abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.

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