Mtr4 is an essential ribonucleic acid (RNA) helicase that plays a central role in RNA processing and degradation as an activator of the nuclear exosome. Mtr4 can carry out its function alone or in the Trf4/Air2/Mtr4 Polyadenylation complex (TRAMP). While recent structures highlight the pathway of RNA through the helicase core, the limited resolution of these studies precludes a detailed description of Mtr4-RNA interactions and how these change with different RNA substrates. The molecular arrangement and mechanism of the TRAMP complex are also unknown. To fill these gaps, we performed an extensive series of hydrogen-deuterium exchange (HDX), binding and activity assays with Mtr4, the TRAMP complex, and different RNA substrates. Our study reveals important RNA interactions that span multiple domains of the Mtr4 helicase, including the Kyprides-Ouzounis-Woese (KOW) motif (or fist) of the Arch domain. Different RNA substrates engage the KOW motif to different extents when they bind Mtr4, and this engagement correlates with RNA binding and unwinding activity. Interactions between the KOW motif and RNA also induce a large conformational change of the arm of the Arch domain. These data support a substrate-specific role for the Arch domain in RNA recognition. We further uncover molecular details about the arrangement and function of TRAMP. We show the path of RNA binding in the Air2-Trf4 sub-complex and identify novel interfaces between Air2-Trf4 and Mtr4. Air2 in fact binds to the Arch domain of Mtr4 and causes Mtr4 to adopt a conformation that resembles an RNA-bound complex. Experiments with RNA and TRAMP also show that RNAbinding by Air2-Trf4 and Mtr4 is similar when the proteins are alone or in TRAMP. This, combined with functional assays, clearly implies competition between the polyadenylation active site of Trf4 and the helicase active site of Mtr4. Consistently, we show that Mtr4 will prevent unregulated RNA polyadenylation by Trf4-Air2. Altogether our data shows that Mtr4 helicase activity is regulated by the RNA substrate, as well as protein binding partners.