• Home
  • Search Results
  • Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII.

Structural and biochemical insights into the DNA-binding mode of MjSpt4p:Spt5 complex at the exit tunnel of RNAPII.

Journal of structural biology (2015-10-04)
Gongrui Guo, Yongxiang Gao, Zhongliang Zhu, Debiao Zhao, Zhihong Liu, Huihao Zhou, Liwen Niu, Maikun Teng
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Imidazole, ACS reagent, ≥99% (titration)
Sigma-Aldrich
Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 10% in H2O
Sigma-Aldrich
Imidazole, ReagentPlus®, 99%
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
Imidazole, for molecular biology, ≥99% (titration)
Sigma-Aldrich
Sodium dodecyl sulfate, ≥99.0% (GC), dust-free pellets
Sigma-Aldrich
Imidazole, puriss. p.a., ≥99.5% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O
Sigma-Aldrich
Sodium dodecyl sulfate, BioXtra, ≥99.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, 92.5-100.5% based on total alkyl sulfate content basis
Sigma-Aldrich
Imidazole, BioUltra, ≥99.5% (GC)
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Imidazole buffer Solution, BioUltra, 1 M in H2O
Supelco
Sodium dodecyl sulfate, dust-free pellets, suitable for electrophoresis, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Sodium chloride, 99.999% trace metals basis
Sigma-Aldrich
Sodium dodecyl sulfate, ≥90%
Sigma-Aldrich
Imidazole, anhydrous, free-flowing, Redi-Dri, ACS reagent, ≥99%