title: Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.
abstract: Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5' and 3' exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3' splice site (3'SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3'SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.
authors.affiliation: Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ohrt
authors.forename: Peter
authors.initials: P
authors.lastname: Odenwälder
authors.forename: Julia
authors.initials: J
authors.lastname: Dannenberg
authors.forename: Mira
authors.initials: M
authors.lastname: Prior
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Warkocki
authors.forename: Jana
authors.initials: J
authors.lastname: Schmitzová
authors.forename: Ramazan
authors.initials: R
authors.lastname: Karaduman
authors.forename: Ingo
authors.initials: I
authors.lastname: Gregor
authors.forename: Jörg
authors.initials: J
authors.lastname: Enderlein
authors.forename: Patrizia
authors.initials: P
authors.lastname: Fabrizio
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D022821:RNA Splice Sites; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D046912:Multiprotein Complexes; D022821:RNA Splice Sites; D012331:RNA, Fungal; D012333:RNA, Messenger; D053487:DEAD-box RNA Helicases
keywords: Prp22; Slu7 Prp18; spliceosome; step 2 catalysis; step 2 factors/Prp16