ABSTRACT
The cytosolic iron-sulfur (Fe-S) assembly (CIA) pathway is required for the insertion of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA replication and repair factors. The molecular mechanisms of client protein recognition and Fe-S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe-S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe-S cluster transfer.
Subject(s)
Iron-Sulfur Proteins/metabolism , Metallochaperones/metabolism , Metalloproteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Line , Crystallography, X-Ray , Drosophila , HEK293 Cells , Humans , Iron-Sulfur Proteins/chemistry , Metallochaperones/chemistry , Metalloproteins/chemistry , Mice , Models, Molecular , Nuclear Proteins/chemistry , Protein Binding , Protein Conformation , Transcription Factors/chemistryABSTRACT
RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.
Subject(s)
Molecular Mimicry , RNA Polymerase II/chemistry , RecQ Helicases/chemistry , Transcription Elongation, Genetic , Transcriptional Elongation Factors/antagonists & inhibitors , Amino Acid Sequence , Binding Sites , Conserved Sequence , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , RNA Polymerase II/metabolism , RNA Polymerase II/ultrastructure , RecQ Helicases/metabolism , RecQ Helicases/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/physiology , Transcriptional Elongation Factors/ultrastructureABSTRACT
Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.
Subject(s)
Epigenetic Repression , RNA Polymerase II/genetics , RNA, Untranslated/genetics , Transcription Factors, TFII/genetics , Animals , Binding Sites/genetics , Cryoelectron Microscopy , HeLa Cells , Heat-Shock Response/genetics , Humans , Image Processing, Computer-Assisted , Mice , Protein Conformation , RNA Polymerase II/chemistry , RNA, Untranslated/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transcription Factors, TFII/chemistry , Up-RegulationABSTRACT
Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPRs), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC.
Subject(s)
Molecular Chaperones/chemistry , Nuclear Pore Complex Proteins/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , RNA/metabolism , Sequence Homology, Amino AcidABSTRACT
Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 A resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.
