biostudies-literatureDenson JMHHS | NIH | National Institute of General Medical SciencesNIGMS NIH HHSHHS | NIH | National Institute of General Medical Sciences (NIGMS)e2414980121https://www.ebi.ac.uk/biostudies/studies/S-EPMC11725892biostudies-literatureUnknown122(1)The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a subcomplex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, theseProceedings of the National Academy of Sciences of the United States of AmericaTRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2.PMC11725892R35 GM133751R15GM148949R01GM117311R35GM133751R01 GM117311R15 GM148949Denson JMThompson KZhang NBall DJohnson SJD'Arcy SfalseTRAMP assembly alters the conformation and RNA binding of Mtr4 and Trf4-Air2.The TRAMP complex contains two enzymatic activities essential for RNA processing upstream of the nuclear exosome. Within TRAMP, RNA is 3' polyadenylated by a subcomplex of Trf4/5 and Air1/2 and unwound 3' to 5' by Mtr4, a DExH helicase. The molecular mechanisms of TRAMP assembly and RNA shuffling between the two TRAMP catalytic sites are poorly understood. Here, we report solution hydrogen-deuterium exchange data with thermodynamic and functional assays to uncover these mechanisms for yeast TRAMP with Trf4 and Air2 homologs. We show that TRAMP assembly constrains RNA-recognition motifs that are peripheral to catalytic sites. These include the Mtr4 Arch and Air2 zinc knuckles 1, 2, and 3. While the Air2 Arch-interacting motif likely constrains the Mtr4 Arch via transient interactions, these2025-01-01T00:00:00Z2025 Jan2026-06-01T16:01:34.305Z2026-04-08T14:03:49.83ZS-EPMC117258923975252610.1073/pnas.2414980121