title: Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach.
abstract: This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3' end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3' end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.
authors.affiliation: Department of Biological Sciences, Wayne State University.
authors.forename: Zuzer
authors.initials: Z
authors.lastname: Dhoondia
authors.affiliation: Department of Biological Sciences, Wayne State University.
authors.forename: Ricci
authors.initials: R
authors.lastname: Tarockoff
authors.affiliation: Department of Biological Sciences, Wayne State University.
authors.forename: Nadra
authors.initials: N
authors.lastname: Alhusini
authors.affiliation: Department of Biological Sciences, Wayne State University.
authors.forename: Scott
authors.initials: S
authors.lastname: Medler
authors.affiliation: Department of Biological Sciences, Wayne State University.
authors.forename: Neha
authors.initials: N
authors.lastname: Agarwal
authors.affiliation: Department of Biological Sciences, Wayne State University; bb2749@wayne.edu.
authors.forename: Athar
authors.initials: A
authors.lastname: Ansari
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D005821:Genetic Techniques; D009154:Mutation; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D004718:Saccharomycetales / Q000235:genetics*; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics
chemical_list: C111825:ASC1 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D016372:RNA, Antisense; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C512638:RNA14 protein, S cerevisiae; C018009:5-bromouridine triphosphate; D019204:GTP-Binding Proteins; D014544:Uridine Triphosphate