title: Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII.
abstract: Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China.
authors.forename: Gongrui
authors.initials: G
authors.lastname: Guo
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China.
authors.forename: Yongxiang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China.
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China.
authors.forename: Debiao
authors.initials: D
authors.lastname: Zhao
authors.affiliation: School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People's Republic of China.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, People's Republic of China. Electronic address: zhuihao@mail.sysu.edu.cn.
authors.forename: Huihao
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China. Electronic address: lwniu@ustc.edu.cn.
authors.forename: Liwen
authors.initials: L
authors.lastname: Niu
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China; Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, People's Republic of China; Innovation Center for Cell Signaling Network, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China. Electronic address: mkteng@ustc.edu.cn.
authors.forename: Maikun
authors.initials: M
authors.lastname: Teng
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D063747:Methanocaldococcus / Q000235:genetics*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D035602:Transcriptional Elongation Factors / Q000378:metabolism*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor
keywords: Archaea; Crystal structure; Fluorescence polarization assays; NusG; Spt5; Transcription factor