RESUMO
Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with â¼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.
Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , Microscopia Crioeletrônica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Complexo Mediador/genética , Iniciação da Transcrição GenéticaRESUMO
Previous structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription have relied on the use of synthetic oligonucleotides, often artificially discontinuous to capture pol II in the initiating state. Here, we report multiple structures of initiation complexes converted de novo from a 33-subunit yeast pre-initiation complex (PIC) through catalytic activities and subsequently stalled at different template positions. We determine that PICs in the initially transcribing complex (ITC) can synthesize a transcript of â¼26 nucleotides before transitioning to an elongation complex (EC) as determined by the loss of general transcription factors (GTFs). Unexpectedly, transition to an EC was greatly accelerated when an ITC encountered a downstream EC stalled at promoter proximal regions and resulted in a collided head-to-end dimeric EC complex. Our structural analysis reveals a dynamic state of TFIIH, the largest of GTFs, in PIC/ITC with distinct functional consequences at multiple steps on the pathway to elongation.
Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Iniciação da Transcrição Genética , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase II/genética , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-Atividade , Fatores de Tempo , Elongação da Transcrição Genética , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismoRESUMO
Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.
Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação ProteicaRESUMO
During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.