ABSTRACT
Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA Polymerase II/metabolism , RNA, Messenger/genetics , Triazines/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , HeLa Cells , Humans , Neoplasms/genetics , Phosphorylation , Roscovitine , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Eukaryotic transcription is regulated by interactions between gene-specific activators and the coactivator complex Mediator. Here we report the NMR structure of the Mediator subunit Med25 (also called Arc92) activator interaction domain (ACID) and analyze the structural and functional interaction of ACID with the archetypical acidic transcription activator VP16. Unlike other known activator targets, ACID forms a seven-stranded ß-barrel framed by three helices. The VP16 subdomains H1 and H2 bind to opposite faces of ACID and cooperate during promoter-dependent activated transcription in a in vitro system. The activator-binding ACID faces are functionally required and conserved among higher eukaryotes. Comparison with published activator structures reveals that the VP16 activation domain uses distinct interaction modes to adapt to unrelated target surfaces and folds that evolved for activator binding.
