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.

Sulfotransferases (STs) catalyze the transfer reaction of the sulfate group from the ubiquitous donor 3 -phosphoadenosine 5 -phosphosulfate (PAPS) to an acceptor group of numerous substrates. This reaction, often referred to as sulfuryl transfer, sulfation, or sulfonation, is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development, and defense. The cytosolic STs sulfate small molecules such as steroids, bioamines, and therapeutic drugs, while the Golgi-membrane counterparts sulfate large molecules including glucosaminylglycans and proteins. We have now solved the X-ray crystal structures of four cytosolic and one membrane ST. All five STs are globular proteins composed of a single ␣/␤ domain with the characteristic five-stranded ␤-sheet. The ␤-sheet constitutes the core of the Paps-binding and catalytic sites. Structural analysis of the PAPS-, PAP-, substrate-, and/or orthovanadate (VO 4 3؊ )-bound enzymes has also revealed the common molecular mechanism of the transfer reaction catalyzed by sulfotransferses. The X-ray crystal structures have opened a new era for the study of sulfotransferases.

The method of crosslinking combined with mass spectrometry is being gradually accepted as a technology enabling detailed structural information on proteins and protein complexes. Intrinsic challenges of the method, which have prevented its widespread use, are being progressively addressed by improvements in mass spectrometry instrumentation capabilities, by the development of new crosslinking reagents, and by the development of specialized software tools for processing of mass spectrometric crosslinking data. This review focuses on recent literature concerning the development of specialized crosslinking reagents and approaches for mass spectrometry-based applications. Critical features of crosslinking reagents for optimum mass spectrometric performance, such as isotopic coding, cleavability, affinity groups, structure of the linkers, and reactive groups, are assessed. Requirements for the design of crosslinking reagents to make them well suited for mass spectrometric detection and analysis are summarized.