Torpedo nuclease Rat1 is insufficient to terminate RNA polymerase II in vitro.
Termination of RNA polymerase (pol) II transcription in vivo requires the 5'-RNA exonuclease Rat1. It was proposed that Rat1 degrades RNA from the 5'-end that is created by transcript cleavage, catches up with elongating pol II, and acts like a Torpedo that removes pol II from DNA. Here we test the Torpedo model in an in vitro system based on bead-coupled pol II elongation complexes (ECs). Recombinant Rat1 complexes with Rai1, and with Rai1 and Rtt103, degrade RNA extending from the EC until they reach the polymerase surface but fail to terminate pol II. Instead, the EC retains an approximately 18-nucleotide RNA that remains with its 3'-end at the active site and can be elongated. Thus, pol II termination apparently requires a factor or several factors in addition to Rat1, Rai1, and Rtt103, post-translational modifications of these factors, or unusual reaction conditions.
Dengl
Stefan
S
Gene Center and Center for Integrated Protein Science Munich, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Cramer
Patrick
P
D001483:Base Sequence; D005095:Exoribonucleases / Q000737:chemistry / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012319:RNA Polymerase II / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013728:Terminator Regions, Genetic; D014961:X-Ray Diffraction
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C473024:Rai1 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C075577:RAT1 protein, S cerevisiae; D012319:RNA Polymerase II; D005095:Exoribonucleases