title: Interaction of RNA polymerase II fork loop 2 with downstream non-template DNA regulates transcription elongation.
abstract: Fork loop 2 is a small semiconservative segment of the larger fork domain in the second largest Rpb2 subunit of RNA polymerase II (Pol II). This flexible loop, juxtaposed at the leading edge of transcription bubble, has been proposed to participate in DNA strand separation, translocation along DNA, and NTP loading to Pol II during elongation. Here we show that the Rpb2 mutant carrying a deletion of the flexible part of the loop is not lethal in yeast. The mutation exhibits no defects in DNA melting and translocation in vitro but confers a moderate decrease of the catalytic activity of the enzyme caused by the impaired sequestration of the NTP substrate in the active center prior to catalysis. In the structural model of the Pol II elongation complex, fork loop 2 directly interacts with an unpaired DNA residue in the non-template DNA strand one nucleotide ahead from the active center (the i+2 position). We showed that elimination of this putative interaction by replacement of the i+2 residue with an abasic site inhibits Pol II activity to the same degree as the deletion of fork loop 2. This replacement has no detectable effect on the activity of the mutant enzyme. We provide direct evidence that interaction of fork loop 2 with the non-template DNA strand facilitates NTP sequestration through interaction with the adjacent segment of the fork domain involved in the active center of Pol II.
authors: NCI-Frederick, National Institutes of Health, Center for Cancer Research, Frederick, Maryland 21702-1201.
authors: Maria L
authors: ML
authors: Kireeva
authors: Gene Transcription and Proteomics Laboratory, Institut de Recherches Cliniques de Montréal and Department of Biochemistry, Université de Montréal, Montréal, Québec, H2W 1R7 Canada.
authors: Céline
authors: C
authors: Domecq
authors: Gene Transcription and Proteomics Laboratory, Institut de Recherches Cliniques de Montréal and Department of Biochemistry, Université de Montréal, Montréal, Québec, H2W 1R7 Canada.
authors: Benoit
authors: B
authors: Coulombe
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors: Zachary F
authors: ZF
authors: Burton
authors: NCI-Frederick, National Institutes of Health, Center for Cancer Research, Frederick, Maryland 21702-1201. Electronic address: kashlevm@mail.nih.gov.
authors: Mikhail
authors: M
authors: Kashlev
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D004247:DNA / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011196:Potassium Permanganate / Q000737:chemistry; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology*; D014158:Transcription, Genetic
chemical_list: D011196:Potassium Permanganate; D004247:DNA; D012319:RNA Polymerase II; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III