title: Poly(A) Signal-Dependent Transcription Termination Occurs through a Conformational Change Mechanism that Does Not Require Cleavage at the Poly(A) Site.
abstract: Transcription termination for genes encoding polyadenylated mRNAs requires a functional poly(A) signal (PAS) in the nascent pre-mRNA. Often called PAS-dependent termination, or PADT, it is widely assumed that the PAS requirement reflects an obligatory poly(A) site cleavage requirement for termination. Cleavage is thought to provide entry for a 5'-to-3' exonuclease that targets RNA polymerase II via the nascent transcript-i.e., the torpedo model. To assess the role of cleavage in PADT, we developed a PADT assay using HeLa nuclear extract. Here we examine the basal mechanism of PADT and show that cleavage at the poly(A) site is not required for PADT. Isolated elongation complexes undergo termination in a PAS-dependent manner when incubated in buffer, in the absence of extract, nucleotides, or cleavage at the poly(A) site. Thus, PADT-proficient complexes undergo a conformational change that triggers termination. PADT is inhibited by α-amanitin, which presumably blocks the required conformational change.
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Isis Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Rigo
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: hgm@chem.ucla.edu.
authors.forename: Harold G
authors.initials: HG
authors.lastname: Martinson
mesh_terms: D053959:Alpha-Amanitin / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011061:Poly A / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D061806:Transcription Termination, Genetic / Q000187:drug effects
chemical_list: D053959:Alpha-Amanitin; D012333:RNA, Messenger; D011061:Poly A