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.

4 0 4 VOLUME 18 NUMBER 4 APRIL 2011 nAture structurAl & moleculAr biologyActivator proteins regulate eukaryotic RNA polymerase II (Pol II) transcription in response to developmental and environmental signals. They bind to the DNA recognition sites of target genes with a sequence-specific DNA-binding domain, and recruit components of the Pol II machinery through a transcriptional activation domain (TAD) 1,2 . Activators have been classified as acidic, glutamine-rich, proline-rich and serine/threonine-rich, depending on the preponderance of amino acids in their TAD 3 . An archetypal acidic activator is the herpes simplex virus protein 16 (VP16), which activates the expression of immediate early viral genes during infection 4-8 . VP16 exerts its activating function through a C-terminal TAD that includes residues 413-490 (refs. 9-13). The VP16 TAD has been intensely used for studying transcriptional activation. Usually, the VP16 TAD is fused with its N terminus to the DNA-binding domain of the yeast transcription factor Gal4. The resulting Gal4-VP16 activator fusion protein stimulates transcription from promoters that contain Gal4-binding sites in the yeast and mammalian transcription systems in vivo 14,15 and in vitro 16,17 . This indicates that basic mechanisms of transcription activation are conserved amongst eukaryotes. The VP16 TAD targets basal Pol II transcription factors, including TFIIA, TFIIB, TFIID, the TFIIH subunit Tfb1/p62 (yeast/human) and the Mediator co-activator [18][19][20][21][22][23] . The VP16 TAD binds the Mediator subunit Med25 (also called Arc92) [24][25][26][27] , which is specific to higher eukaryotes. Med25 consists of two domains, the activator interaction domain (ACID) 24 that binds the VP16 TAD, and a 'von Willebrand factor type A' domain that anchors Med25 to Mediator 24 . Mediator generally conveys regulatory information by forming a bridge between activators and the basal Pol II machinery 28,29 . Whereas information on the core Mediator structure is emerging, structural information about its more peripheral activator-binding domains is limited to the KIX domain in subunit Med15 (or Arc105) 30,31 .The VP16 TAD is intrinsically flexible, whereas the VP16 core domain (residues 49-402) forms a stable structure 23,[32][33][34] . The TAD contains two functional subdomains, H1 (residues 410-452) and H2 (residues 453-490), which activate transcription independently 35,36 . Both H1 and H2 contain acidic amino acids, but specific bulky hydrophobic and aromatic residues are required for their function [36][37][38][39] . H2 has been proposed to adopt an α-helical conformation when bound to TFIIB 40 . NMR analysis revealed a nine-residue amphipathic α-helix in H2 that docks onto a PH fold in Tfb1 21 . On the basis of these and other results it is assumed that acidic TADs are flexible in their free state, but become transiently structured upon interaction with their target proteins.Here we report the NMR structure of the Mediator Med25 ACID and analyze its structural and functional interaction wi...