Structures of dengue virus RNA replicase complexes.

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

Cryo-EM structure and biochemical analyses of the nucleosome containing the cancer-associated histone H3 mutation E97K.

The Lys mutation of the canonical histone H3.1 Glu97 residue (H3E97K) is found in cancer cells. Previous biochemical analyses revealed that the nucleosome containing the H3E97K mutation is extremely unstable as compared to the wild-type nucleosome. However, the mechanism by which the H3E97K mutation causes nucleosome instability has not been clarified yet. In the present study, the cryo-electron microscopy structure of the nucleosome containing the H3E97K mutation revealed that the entry/exit DNA regions of the H3E97K nucleosome are disordered, probably by detachment of the nucleosomal DNA from the H3 N-terminal regions. This may change the intra-molecular amino acid interactions with the replaced H3 Lys97 residue, inducing structural distortion around the mutated position in the nucleosome. Consistent with the nucleosomal DNA end flexibility and the nucleosome instability, the H3E97K mutation exhibited reduced binding of linker histone H1 to the nucleosome, defective activation of PRC2 (the essential methyltransferase for facultative heterochromatin formation) with a poly-nucleosome, and enhanced nucleosome transcription by RNA polymerase II.

Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Cryo-EM structures of RNA polymerase II-nucleosome complexes rewrapping transcribed DNA.

RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.

Crystal structure of RNA polymerase II from Komagataella pastoris.

RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, the crystal structure of Pol II from the methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While the structure is highly homologous to that of Pol II from the budding yeast Saccharomyces cerevisiae, the stalk and clamp modules of the K. pastoris Pol II displayed large inward rotations, closing the central cleft to a greater extent than in the known S. cerevisiae Pol II structures. The conformational differences reflect the inherent flexibilities of the stalk and the clamp, as additional low-resolution structures of K. pastoris Pol II in different crystal forms revealed diverse stalk and clamp orientations. Comparisons with other eukaryotic/archaeal RNA polymerase structures in the Protein Data Bank revealed the distributions of the stalk and clamp orientations. The conformational plasticity should be essential for transcriptional functions and binding various regulatory factors.

Crystal structure of nanoKAZ: The mutated 19 kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine.

The 19 kDa protein (KAZ) of Oplophorus luciferase is a catalytic component, that oxidizes coelenterazine (a luciferin) with molecular oxygen to emit light. The crystal structure of the mutated 19 kDa protein (nanoKAZ) was determined at 1.71 Å resolution. The structure consists of 11 antiparallel ß-strands forming a ß-barrel that is capped by 4 short α-helices. The structure of nanoKAZ is similar to those of fatty acid-binding proteins (FABPs), even though the amino acid sequence similarity was very low between them. The coelenterazine-binding site and the catalytic site for the luminescence reaction might be in a central cavity of the ß-barrel structure.

Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) assemble via distinct modes.

Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) have a large, heavily glycosylated luminal domain composed of two subdomains, and are the most abundant protein components in lysosome membranes. LAMP-1 and LAMP-2 have distinct functions, and the presence of both proteins together is required for the essential regulation of autophagy to avoid embryonic lethality. However, the structural aspects of LAMP-1 and LAMP-2 have not been elucidated. In the present study, we demonstrated that the subdomains of LAMP-1 and LAMP-2 adopt the unique ß-prism fold, similar to the domain structure of the dendritic cell-specific-LAMP (DC-LAMP, LAMP-3), confirming the conserved aspect of this family of lysosome-associated membrane proteins. Furthermore, we evaluated the effects of the N-domain truncation of LAMP-1 or LAMP-2 on the assembly of LAMPs, based on immunoprecipitation experiments. We found that the N-domain of LAMP-1 is necessary, whereas that of LAMP-2 is repressive, for the organization of a multimeric assembly of LAMPs. Accordingly, the present study suggests for the first time that the assembly modes of LAMP-1 and LAMP-2 are different, which may underlie their distinct functions.

Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris.

We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.

Structural perspectives on transcription in chromatin.

In eukaryotes, all genetic processes take place in the cell nucleus, where DNA is packaged as chromatin in 'beads-on-a-string' nucleosome arrays. RNA polymerase II (RNAPII) transcribes protein-coding and many non-coding genes in this chromatin environment. RNAPII elongates RNA while passing through multiple nucleosomes and maintaining the integrity of the chromatin structure. Recent structural studies have shed light on the detailed mechanisms of this process, including how transcribing RNAPII progresses through a nucleosome and reassembles it afterwards, and how transcription elongation factors, chromatin remodelers, and histone chaperones participate in these processes. Other studies have also illuminated the crucial role of nucleosomes in preinitiation complex assembly and transcription initiation. In this review we outline these advances and discuss future perspectives.

Structural Transition of the Nucleosome during Transcription Elongation.

In eukaryotes, genomic DNA is tightly wrapped in chromatin. The nucleosome is a basic unit of chromatin, but acts as a barrier to transcription. To overcome this impediment, the RNA polymerase II elongation complex disassembles the nucleosome during transcription elongation. After the RNA polymerase II passage, the nucleosome is rebuilt by transcription-coupled nucleosome reassembly. Nucleosome disassembly-reassembly processes play a central role in preserving epigenetic information, thus ensuring transcriptional fidelity. The histone chaperone FACT performs key functions in nucleosome disassembly, maintenance, and reassembly during transcription in chromatin. Recent structural studies of transcribing RNA polymerase II complexed with nucleosomes have provided structural insights into transcription elongation on chromatin. Here, we review the structural transitions of the nucleosome during transcription.

Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus.

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The ß-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Structural Basis of Damaged Nucleotide Recognition by Transcribing RNA Polymerase II in the Nucleosome.

In transcription-coupled repair (TCR), transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits TCR proteins to the damaged site. However, the mechanism by which RNAPII recognizes a DNA lesion in the nucleosome remains enigmatic. In the present study, we inserted an apurinic/apyrimidinic DNA lesion analogue, tetrahydrofuran (THF), in the nucleosomal DNA, where RNAPII stalls at the SHL(-4), SHL(-3.5), and SHL(-3) positions, and determined the structures of these complexes by cryo-electron microscopy. In the RNAPII-nucleosome complex stalled at SHL(-3.5), the nucleosome orientation relative to RNAPII is quite different from those in the SHL(-4) and SHL(-3) complexes, which have nucleosome orientations similar to naturally paused RNAPII-nucleosome complexes. Furthermore, we found that an essential TCR protein, Rad26 (CSB), enhances the RNAPII processivity, and consequently augments the DNA damage recognition efficiency of RNAPII in the nucleosome. The cryo-EM structure of the Rad26-RNAPII-nucleosome complex revealed that Rad26 binds to the stalled RNAPII through a novel interface, which is completely different from those previously reported. These structures may provide important information to understand the mechanism by which RNAPII recognizes the nucleosomal DNA lesion and recruits TCR proteins to the stalled RNAPII on the nucleosome.

Cryo-EM and biochemical analyses of the nucleosome containing the human histone H3 variant H3.8.

Histone H3.8 is a non-allelic human histone H3 variant derived from H3.3. H3.8 reportedly forms an unstable nucleosome, but its structure and biochemical characteristics have not been revealed yet. In the present study, we reconstituted the nucleosome containing H3.8. Consistent with previous results, the H3.8 nucleosome is thermally unstable as compared to the H3.3 nucleosome. The entry/exit DNA regions of the H3.8 nucleosome are more accessible to micrococcal nuclease than those of the H3.3 nucleosome. Nucleosome transcription assays revealed that the RNA polymerase II (RNAPII) pausing around the superhelical location (SHL) -1 position, which is about 60 base pairs from the nucleosomal DNA entry site, is drastically alleviated. On the other hand, the RNAPII pausing around the SHL(-5) position, which is about 20 base pairs from the nucleosomal DNA entry site, is substantially increased. The cryo-electron microscopy structure of the H3.8 nucleosome explains the mechanisms of the enhanced accessibility of the entry/exit DNA regions, reduced thermal stability and altered RNAPII transcription profile.

H2A Ubiquitination Alters H3-tail Dynamics on Linker-DNA to Enhance H3K27 Methylation.

Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2-AEBP2-JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2-AEBP2-JARID2.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes.

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Structural basis of nucleosome disassembly and reassembly by RNAPII elongation complex with FACT.

During gene transcription, RNA polymerase II (RNAPII) traverses nucleosomes in chromatin, but the mechanism has remained elusive. Using cryo-electron microscopy, we obtained structures of the RNAPII elongation complex (EC) passing through a nucleosome in the presence of the transcription elongation factors Spt6, Spn1, Elf1, Spt4/5, and Paf1C and the histone chaperone FACT (facilitates chromatin transcription). The structures show snapshots of EC progression on DNA mediating downstream nucleosome disassembly, followed by its reassembly upstream of the EC, which is facilitated by FACT. FACT dynamically adapts to successively occurring subnucleosome intermediates, forming an interface with the EC. Spt6, Spt4/5, and Paf1C form a "cradle" at the EC DNA-exit site and support the upstream nucleosome reassembly. These structures explain the mechanism by which the EC traverses nucleosomes while maintaining the chromatin structure and epigenetic information.

Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1.

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.

Structural and biochemical analyses of the nucleosome containing Komagataella pastoris histones.

Komagataella pastoris is a methylotrophic yeast that is commonly used as a host cell for protein production. In the present study, we reconstituted the nucleosome with K. pastoris histones and determined the structure of the nucleosome core particle by cryogenic electron microscopy. In the K. pastoris nucleosome, the histones form an octamer and the DNA is left-handedly wrapped around it. Micrococcal nuclease assays revealed that the DNA ends of the K. pastoris nucleosome are somewhat more accessible, as compared with those of the human nucleosome. In vitro transcription assays demonstrated that the K. pastoris nucleosome is transcribed by the K. pastoris RNA polymerase II (RNAPII) more efficiently than the human nucleosome, while the RNAPII pausing positions of the K. pastoris nucleosome are the same as those of the human nucleosome. These results suggested that the DNA end flexibility may enhance the transcription efficiency in the nucleosome but minimally affect the nucleosomal pausing positions of RNAPII.