Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4-Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

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...