title: In vitro transcription of a torsionally constrained template.
abstract: RNA polymerase (RNAP) and the DNA template must rotate relative to each other during transcription elongation. In the cell, however, the components of the transcription apparatus may be subject to rotary constraints. For instance, the DNA is divided into topological domains that are delineated by rotary locked boundaries. Furthermore, RNAPs may be located in factories or attached to matrix sites limiting or prohibiting rotation. Indeed, the nascent RNA alone has been implicated in rotary constraining RNAP. Here we have investigated the consequences of rotary constraints during transcription of torsionally constrained DNA by free RNAP. We asked whether or not a newly synthesized RNA chain would limit transcription elongation. For this purpose we developed a method to immobilize covalently closed circular DNA to streptavidin-coated beads via a peptide nucleic acid (PNA)-biotin conjugate in principle mimicking a SAR/MAR attachment. We used this construct as a torsionally constrained template for transcription of the beta-lactamase gene by Escherichia coli RNAP and found that RNA synthesis displays similar characteristics in terms of rate of elongation whether or not the template is torsionally constrained. We conclude that transcription of a natural bacterial gene may proceed with high efficiency despite the fact that newly synthesized RNA is entangled around the template in the narrow confines of torsionally constrained supercoiled DNA.
authors.affiliation: Center for Biomolecular Recognition, IMBG, Laboratory B, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bentin
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
mesh_terms: D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial / Q000235:genetics; D007700:Kinetics; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012399:Rotation; D019809:Streptavidin / Q000378:metabolism; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D020135:Peptide Nucleic Acids; C051802:triplex DNA; D012313:RNA; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D012321:DNA-Directed RNA Polymerases; D001618:beta-Lactamases