Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif

Nucleic Acids Res. 2010 Jul;38(12):4040-51. doi: 10.1093/nar/gkq135. Epub 2010 Mar 2.

Abstract

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism
  • Binding Sites
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Chromosomal Proteins, Non-Histone / metabolism
  • Conserved Sequence
  • Crystallography, X-Ray
  • DNA-Directed RNA Polymerases / metabolism*
  • Hydrophobic and Hydrophilic Interactions
  • Methanococcus
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Transcription, Genetic*
  • Transcriptional Elongation Factors / chemistry*
  • Transcriptional Elongation Factors / metabolism

Substances

  • Archaeal Proteins
  • Chromosomal Proteins, Non-Histone
  • Transcriptional Elongation Factors
  • SPT5 transcriptional elongation factor
  • DNA-Directed RNA Polymerases