Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.
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.
Hirtreiter
Angela
A
Division of Biosciences, University College London, Institute for Structural and Molecular Biology, Darwin Building, Gower Street, London WC1E 6BT, UK.
Damsma
Gerke E
GE
Cheung
Alan C M
AC
Klose
Daniel
D
Grohmann
Dina
D
Vojnic
Erika
E
Martin
Andrew C R
AC
Cramer
Patrick
P
Werner
Finn
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D017017:Methanococcus; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry* / Q000378:metabolism
D019843:Archaeal Proteins; D002868:Chromosomal Proteins, Non-Histone; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D012321:DNA-Directed RNA Polymerases