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It is a qualitative research study, descriptive-exploratory in nature, which aims to verify the benefits from the use of toys during nursing care to hospitalized children. Ten subjects participated in the study: three children and seven mothers of hospitalized children. Data were collected between May and July, 2008 by means of specific instruments for each age group and further organized in thematic categories: the use of toys to lessen hospitalization stress; toys facilitating understanding and acceptance of procedures; and the experience of using toys and hospitalization process. The results show that the use of toys is an excellent nursing resource to render care to admitted children. The features of the toys facilitated communication, participation, acceptance of procedure and child motivation, what enabled them to keep their individuality, lessen the stress and the possibility to implement children's and families' non-traumatic care. Source

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In every living cell, a large macromolecular complex called the ribosome is responsible for translating messenger RNA into amino-acid chains in the cytoplasm. A mature ribosome contains about 80 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs). Yet the construction of a ribosome is mediated by many more proteins and RNA molecules within large dynamic pre-ribosomal complexes. Writing in Cell, Kornprobst et al.1 report that they have exploited advances in cryo-electron microscopy2 to resolve the structure of the earliest pre-ribosome, the 90S, to a near-atomic resolution of between 4 and 7 ångströms. The structure reveals, for the first time and in stunning detail, the arrangement of and interactions between many proteins that have been implicated in ribosome assembly, shedding light on a crucial step in early ribosome formation. In 1967, it was discovered3 that, in eukaryotic organisms (those whose cells carry a nucleus), a long RNA transcript called the pre-rRNA undergoes processing in a nuclear compartment, the nucleolus, to produce three of the four rRNAs found in the mature ribosome. An analysis4 later that year of ribosomes isolated from human nuclei, and a comparison5 of cytoplasmic and nuclear ribosomes in 1972, revealed that nuclear ribosomes contain many more proteins than do their cytoplasmic counterparts. These extra proteins were hypothesized to help process the pre-rRNA. Since then, the steps of pre-rRNA processing have been established and most of the extra proteins (now called ribosome biogenesis factors) have been identified, thanks to advances in biochemistry and mass spectrometry. During its transcription, the long pre-rRNA is assembled with r-proteins, ribosome biogenesis factors and small nucleolar RNAs to form a large 90S pre-ribosome. Following the first stage of pre-rRNA processing, the complex splits into two pre-ribosomes, dubbed pre-40S and pre-60S, which are eventually exported to the cytoplasm where they undergo further maturation steps and then join as 40S and 60S subunits to form the mature ribosome. Along with the identities of the biogenesis factors came the realization that they numbered a vast 200 to 300 in eukaryotes6, 7. In the yeast Saccharomyces cerevisiae, the 90S pre-ribosome alone contains about 70 ribosome biogenesis factors — almost as many as the number of proteins in a mature ribosome6. Hence, a recurring question in the field is: why does ribosome production require so many accessory proteins? By resolving the structure of the 90S pre-ribosome in the yeast Chaetomium thermophilum, Kornprobst et al. provide an answer to this question. The authors identified features in their structure by fitting data from previous biochemical and genetic studies (including X-ray structures of several proteins, predicted protein-domain structures and known protein–protein and protein–pre-rRNA interactions) to determine where different proteins and RNAs are located in the 90S complex. The requirement for so many extra proteins is explained by the authors' observation that many accessory proteins are arranged around the folded pre-rRNA molecule in previously defined8 multi-protein complexes called UTP-A, UTP-B and UTP-C. Of these, UTP-A and UTP-B form a scaffold, within which the newly transcribed pre-rRNA is encased and so can be securely processed, modified and assembled with r-proteins (Fig. 1). The role of this scaffold is reminiscent of the way in which chaperone proteins aid folding of other proteins — a common process that prevents aggregation of proteins into non-functional structures. But although chaperone-mediated protein folding has been long established9, the idea of chaperone moulds is new to RNA biology. The 90S chaperone mould also includes the small nucleolar ribonucleoprotein complex U3 — an RNA–protein complex that has known roles in pre-rRNA processing and folding10, 11. Kornprobst et al. showed that one half of U3 spans the outer body of the 90S complex in a scaffold-like arrangement, whereas the other half is buried deep within the 90S, presumably interacting with the pre-rRNA. This part of U3 is associated with a region at the end of the pre-rRNA called the 5′ external transcribed spacer (5′-ETS), and the authors demonstrated that cleavage of this spacer from the pre-rRNA is crucial for the separation of the processed 90S pre-rRNAs into pre-40S and pre-60S complexes, and the progression of ribosome production. Kornprobst and colleagues also identified the position of the pre-18S rRNA (which will become the rRNA component of the 40S subunit) in their structure. When comparing the pre-18S structure with that of the mature 18S rRNA, the authors observed that the molecule underwent progressive folding, beginning in the domains closest to the site where transcription began. In the 90S, these regions were folded to resemble the mature 18S, whereas domains farther from the transcriptional start site were seemingly still in transitory states. This observation fits well with a previous model6 of hierarchical rRNA assembly. Kornprobst and colleagues have visualized in detail what, until now, has been seen through electron microscopy only as small black balls on strings of pre-rRNA. Holding a magnifying glass to the early steps of ribosome biogenesis, the authors have finally revealed a role for the multitude of ribosome biogenesis factors as a chaperone mould that provides a secure environment for the processing and folding of pre-rRNA. The 90S pre-ribosome contains the entire rRNA precursor, which includes several transcribed spacer sequences that will be cleaved away, and sequences that will give rise to the rRNAs of the 60S ribosomal subunit. However, Kornprobst et al. focused on only the rRNA region and the proteins that give rise to the 40S subunit. As such, many questions about 60S formation remain unanswered — for instance, whether a separate chaperone-like mould encases these other regions of the pre-rRNA. There are several structures visible in 90S that have not yet been identified. In years to come, it will be interesting to index these features and further unravel the role of the UTP-C complex and other proteins in 90S pre-rRNA maturation. Using the technical advances highlighted in the current study, we can hope to shed more light on the dynamic and multi-tiered process that is ribosome formation.

The number of times each of the experiments described in this work was performed and the raw western blots used in the figures are shown in the Supplementary Information file. The investigators were not blinded to allocation during experiments and outcome assessment. In vitro methylation assays (30 μl) containing 100 μM of biotinylated 13-mer CTD peptides centred around R1810 or R1603, 0.1–2 μM PRMT5–WDR77 complex, 100 μM tritiated SAM (PerkinElmer, catalogue number NET155V250UC), 20 mM Tris-HCl pH8, 0.01% TritonX-100 (Sigma catalogue number T8532), and 5 mM DTT were incubated at room temperature for 2 h. The biotinylated peptides were then precipitated with 20 μl streptavidin–agarose (Invitrogen catalogue number SA100), washed with buffer (above), mixed with 5 ml ScintiVerse BD Cocktail (Fisher Chemical, catalogue number SX18-4), and counted using a Beckman Liquid Scintillation Counter (LS 6500). Other similar reactions contained recombinant GST proteins (GST alone, GST-N-CTD or GST-C-CTD), 0.1–2 μM PRMT5–WDR77 and 100 μM tritiated SAM. These reactions were precipitated with glutathione beads (Invitrogen catalogue number G2879), and the beads were washed and eluted with 500 μl of 20 mM L-glutathione (pH 8) for counting. FITC- and biotin-labelled CTD peptides containing R1603 (SPAYEPRSPGGYT) and R1810 (YSPSSPRYTPQSP) were prepared on a Prelude peptide synthesizer (Protein Technologies, Tucson Arizona) using Fmoc (9-fluorenyl methoxycarbonyl) solid-phase chemistry. Dimethyl arginine derivatives were prepared using Fmoc-SDMA(Boc)2-ONa or Fmoc-ADMA (Pbf)-OH reagents (Novabiochem, Germany). Peptides were purified using C18 reverse-phase HPLC and authenticated using mass spectrometry. Constructs for GST recombinant protein expression (GST-N-CTD: contains repeats 1–29; or GST-C-CTD: contains repeats 24–52) were expressed in BL21 bacteria and purified following the standard glutathione bead purification protocol. Bacterial expression constructs and purified Tudor domains from TDRD3, SMN, SPF30, TDRD1, TDRD2, TDRD9 and TDRD11 (also known as SND1) were described previously48. Fluorescence polarization assays were carried out as described before48. The buffer used in the fluorescence polarization assay was 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT and 0.01% Triton X-100. An excitation wavelength of 485 nm and an emission wavelength of 528 nm were used. The data were obtained at 25 °C and corrected by subtracting the label-free peptide background. The data were collected by the Synergy 2 (BioTec, USA) fluorescence polarization program and were fitted to a one-site binding model using Origin 7 (MicroCal). The K values are from the average of three measurements. For isothermal titration calorimetry, the concentrated protein was diluted in 20 mM Tris, pH 7.5, 150 mM NaCl. The lyophilized peptides were dissolved in the same buffer and pH was adjusted by adding NaOH. Peptide concentrations were estimated from the molecular weight. All the measurements were performed at 25 °C, using a VP-ITC microcalorimeter. Protein with a concentration of 50 μM was placed in the cell chamber, and the peptides with a concentration of 1 mM in syringe were injected in 25 successive injections with a spacing of 180 s and a reference power of 13 mcal s−1. Data were fitted using the single-site binding model within the Origin software package (MicroCal). There was no evidence of mycoplasma contamination of the cell lines used in this work as judged by staining of fixed cells with DAPI. Raji cells were cultured in RPMI (SLRI media facility) plus 10% FBS (Sigma catalogue number F1051) and 1% glutamate, and stably transduced cells were maintained with 500 μg ml−1 G418 (Gibco catalogue number 11811031). HEK293 cells were grown in DMEM (SLRI media facility) plus FBS (Sigma catalogue number F1051), and stably transduced cell lines were maintained with 2 μg ml−1 puromycin (Sigma catalogue number p8833). shRNAs in lentivirus vectors were used to stably transduce cell lines using an established protocol49. siRNA knockdowns for HEK293 cells were performed with 50 nM SMARTpool siRNAs with PepMute siRNA transfection reagent (SigmaGen Laboratory catalogue number SL100566) for 3 days. SMARTpool On-Target plus siRNAs against human PRMT5 (catalogue number L-015817) and SMN (catalogue number L-011108) were purchased from Thermo Scientific. For CRISPR-mediated gene knockouts, CRISPR/Cas9 plasmids (pCMV-Cas9-GFP) were purchased from Sigma-Aldrich which express scrambled guide RNA, or guide RNA that targets the SMN1 gene. 2 μg of the plasmids were transfected into HEK293 cells, and 1 day after transfection, cells were sorted by BD FACSAria flow cytometry (Donnelly Centre, University of Toronto) and single GFP-positive cells were plated into a 48-well plate. The expression levels of SMN in each clone were detected by western blotting. Raji cells with stable expression of HA-tagged wild type or POLR2A (R1810A) constructs were generated by electroporation (10 μg of plasmid DNA per 107 cells), followed by selection and maintenance with G418 (0.5 mg ml−1). α-amanitin treatment was carried out with 2 μg ml−1 α-amanitin for 3 days for Co-IP and ChIP experiments involving HA-tagged wild-type or POLR2A (R1810A). The transfection of the GFP-HB transgene for R-loop detection into HEK293 cell lines was performed with the FuGENE Transfection reagent (Roche, catalogue number E269A). SMA disease relevant and control fibroblast and B-lymphocyte cell lines were obtained from the Coriell Institute (Family 553: GM03813, GM03814, GM03815; Family 3042: GM23686, GM23687, GM23688), and were grown in conditions as instructed by the Coriell Institute. The cells were collected and fixed for RNAP II ChIP and R-loop DIP. IP was performed with RIPA buffer (140 mM NaCl, 10 mM Tris pH 7.6–8.0, 1% Triton, 0.1% sodium deoxycholate, 1 mM EDTA) containing protease inhibitors (Roche catalogue number 05892791001) and benzonase (Sigma E1014). 107 to 2 × 107 cells were lysed on ice for 25 min by vortexing and forcing them through a 27 gauge needle. After centrifuging at 13,000 r.p.m. for 10 min at 4 °C, the supernatant was incubated with 25 μl (1:10 dilution) of protein G beads (Invitrogen catalogue numbers 10-1243 and10003D) and 1–2 μg of antibodies for 4 h to overnight. The samples were washed 3 times with RIPA buffer and boiled in SDS gel sample buffer. To detect R1810me2s or R1810me2a modifications on POLR2A, alkaline phosphatase (Roche catalogue number 10108138001) treatment (5 μl) at 37 °C for 30 min was performed for POLR2A immunoprecipitated samples before boiling. Samples were run using 7.5–10% SDS–PAGE and transferred to PVDF membranes (Bio-Rad catalogue number 162-0177) using a trans-blot semi-dry electrophoretic transfer Cell (BioRad catalogue number 170-3940). Primary antibodies were used at 1:250 to 1:1,000 dilutions for incubation overnight, and horseradish peroxidase-conjugated goat anti-mouse, anti -rabbit, or anti-rat secondary antibodies were used at 1:10,000 (Dako catalogue number P0450). Blots were developed using SuperSignal West Pico or Femto (Thermo Scientific catalogue numbers 34079 and 34094). Blots were quantified using ImageJ software. A Hoefer slot blot system (Fisher Scientific catalogue number 11509543) was used to assay R1810me2s antibody specificities following the manufacturer’s protocol. ChIP was performed using the EZ-ChIP A chromatin immunoprecipitation kit (Millipore catalogue number 17-371) or similar homemade solutions according to the manufacturer’s instructions. Antibodies were used in the 1–2 μg range, and IgG was used as a background control. DIP was performed according to ref. 50 with minor modifications. DIP was performed following the ChIP protocol except that, after the nuclear lysis and sonication, genomic DNA was de-crosslinked in ChIP elution buffer containing 5 M NaCl at 65 °C overnight. DNA was purified with the Qiaex II kit (Qiagen catalogue number 20021) for PCR product purification and eluted in water. DIP was carried out overnight with 25 μl of Dynabeads protein G beads (Invitrogen catalogue number 100-03D) and 1 μg of antibody purified from the S9.6 hybridoma cell line51 that recognizes RNA–DNA hybrids. Immunoprecipitated and input DNAs were used as templates for qPCR. DIP RNase-sensitivity analysis was carried out by adding 50 U of RNase H (Invitrogen catalogue number 18021-014) in 10× RNase H buffer (75 mM KCl, 50 mM Tris pH 8.3, 3 mM MgCl , 10 mM DTT) with 4% glycerol and 20 μg ml−1 BSA before immunoprecipitation. The RNase H treatment was performed for 2 h at 37 °C. For comparing POLR2A ChIP and S9.6 DIP signals on the ACTB gene, wild-type or control knockdown signals were normalized to 1, and the R1810A mutant or knockdown samples were adjusted such that the ratio for the intron 3 (1671) position was set to 1. Similarly, for the GAPDH gene, the ratio for the intron 5 (2436) position was set to 1. ChIP data for senataxin and SMN were expressed as ratios to the ChIP data for POLR2A. Error bars represent biological replicates, except where indicated otherwise. NRO was performed according to Skourti-Stathaki et al. with modifications19, 21. Approximately 107 cells were incubated on ice in swelling buffer (10 mM Tris-Cl pH 7.5, 2 mM MgCl , 3 mM CaCl ) for 5 min, and were pelleted. Pellets were resuspended in 1 ml lysis buffer (swelling buffer containing 0.5% NP40, 10% glycerol, and 2 U ml−1 RNaseOUT (Invitrogen catalogue number 10777-019)) and pipetted for lysis, followed by centrifugation. The pellet was resuspended in 1 ml freezing buffer (50 mM Tris-Cl pH 8.3, 40% glycerol, 5 mM MgCl , 0.1 mM EDTA). Reactions contained 100 μl resuspended nuclei, 100 μl reaction buffer (40 mM Tris pH 7.9, 300 mM KCl, 10 mM MgCl , 40% glycerol, 2 mM DTT), 500 μM rNTPs (ATP, CTP, GTP) (GE catalogue number 27-2025-01), including 125 μM UTP as a negative control or Br-UTP (Invitrogen catalogue number B21551) for 30 min at 30 °C. 3 μl BrdU antibody (Sigma catalogue number B8434) was pre-conjugated to 30 μl Dynabeads protein G beads with 10 μg tRNA (Invitrogen catalogue number115401) as block in 100-RSB buffer (10 mM Tris pH 7.4, 100 mM NaCl, 2.5 mM MgCl , 0.4% Triton X-100) for 2 h at 4 °C. The RNA was extracted using TRIzol (Invitrogen catalogue number 15596-026) and was heat fragmented at 95 °C for 8 min. RNA was then mixed with beads and BrdU antibody for 2 h at 4 °C in 500 μl 100-RSB buffer, 100 U ml−1 RNase OUT, 400 U ml−1 DNase I (Invitrogen catalogue number 18047-019). Immunoprecipitated RNA was washed three times with 100-RSB buffer. Primers used in ChIP, DIP, and NRO are listed here19, 52. Chromatin immunoprecipitation was performed as before53. In brief, 107 to 108 cells were crosslinked for 10 min in 1% formaldehyde. Lysates were sonicated to a DNA fragment length range of 200–300 bp using a Bioruptor (Diagenode). RNAP II was immunoprecipitated with 2 μg of antibodies and Dynabeads Protein G (Invitrogen). Subsequently, crosslinks were reversed at 65 °C overnight and bound DNA fragments were purified (EZ-10 spin column PCR product purification kit, Bio Basic). Sequencing libraries were constructed using the TruSeq ChIP sample prep kit (Illumina) according the manufacturer’s instructions. Libraries were sequenced (single-end reads) on the Illumina HiSeq 2500 to a minimum depth of 30 million reads, obtaining at least 10 million unique reads per sample. ChIP-seq analysis was performed chiefly as before54. For ChIP-seq, reads in FASTQ format were mapped to the human genome (hg19) using Bowtie 2 (ref. 55) with local alignment, duplicate reads were removed, and reads were extended to 300 bp. Pileups—the number of fragments overlapping each genomic bp—were calculated, and were normalized by million mappable reads in the ChIP-seq library. Normalized pileups from different replicates were then averaged to create FPM (fragments per million reads). Data for ChIP-seq analyses have been deposited in GEO with the accession code GSE73379. RNA was used for cDNA synthesis with the SuperScript VILO Kit (Invitrogen catalogue number 11754). PCR was performed using the Phusion-high fidelity PCR kit (Thermo Scientific catalogue number F-553S), and qPCR was performed with Fast SYBR Green Master qPCR mix, using the Applied Biosystems 7300 real time PCR System (catalogue number 4406984). qPCR consisted of 40 cycles of 95 °C for 15 s and 55 °C for 30 s, and a final cycle (95 °C for 15 s and then 60 °C) generated a dissociation curve. Input DNA or RNA reverse transcribed into cDNA were used to calculate the per cent enrichment in the immunoprecipitated samples. Anti-CTD R1810me2s antibody was raised in rabbits using a KLH-conjugated CTD peptide from POLR2A (amino acids 1806–1813) that carried an R1810me2s modification. KLH conjugation was performed using an N-terminal cysteine residue (Cedarlane). R1810me2s-specific antibodies were enriched by flowing the serum through a column containing an R1810me0 peptide conjugated to SulfoLink Coupling Resin (Thermo Scientific catalogue number 20401). GST fusion constructs containing CTD N-terminal repeats 1–29 and C-terminal repeats 33–52 were provided by J. Manley56. Flp-in T-REx GFP-HB fusion construct that contains the R-loop binding domain of RNase H was provided by A. Aguilera22. The ORFs for POLR2D, TDRD3, PRMT5, and SMN came from the plasmid collection at Harvard. CMV promoter-driven Flag-tagged TDRD3, GFP, and PRMT5 constructs for HEK293 cell culture were generated using the MAPLE system as previously described49. α-amanitin-resistant wild-type and R1810A mutant POLR2A constructs were kindly provided by D. Eick7. RNAP II R1810 me2a antibody was provided by D. Reinberg7. We obtained the POLR2A pSer2 and pSer5 antibodies from the Eick laboratory (S2P: 3E10; S5P: 3E8). We obtained the S9.6 antibody for R-loop DIP from S. Leppla. 8WG16 monoclonal antibody against unphosphorylated CTD repeats of POLR2A was prepared in our laboratory. Commercial antibodies were as follows: Y12 (Abcam monoclonal antibody catalogue number ab3138); Sym10 (Millipore polyclonal antibody catalogue number 07-412); Asym24 (Millipore polyclonal antibody catalogue number 07-414); TDRD3 (Santa Cruz polyclonal antibody catalogue number C-20); HA (Sigma monoclonal antibody catalogue number H9658); PRMT5 (Upstate polyclonal antibody catalogue number C7-405, Santa Cruz monoclonal antibody catalogue number sc-22132); SMN (Santa Cruz polyclonal antibody catalogue number H-195); SETX (for ChIP and IP (Novus Biologicals polyclonal antibody catalogue number NB100-57543) and for western blots (Bethyl Lab polyclonal antibody catalogue number A301-104A)); XRN2 (Santa Cruz polyclonal antibody catalogue number sc-99237); POLR2A N20 (Santa Cruz polyclonal antibody catalogue number sc-899); POLR2A 4H8 (Abcam monoclonal antibody catalogue number ab5408); POLR2A H224 (Santa Cruz polyclonal antibody catalogue number sc9001); γH2AX (Millipore, catalogue number 05–636); H2AX (Millipore, catalogue number 07–627); tubulin (Sigma monoclonal antibody catalogue number T8328); Flag (Sigma monoclonal antibody catalogue number F1804); GFP (Abcam polyclonal antibody catalogue number 290); IgG negative controls for ChIP and IP (Millipore polyclonal antibody catalogue number 12–370). α-amanitin was purchased from Sigma (catalogue number 23109-05-9).

Kostek R.,UTP
Key Engineering Materials | Year: 2014

This article presents a non-linear model of deep groove ball bearing and results of simulation. Vibrations of this bearing are studied for a wide range of clearance. An evolution from periodic to chaotic vibrations is visible due to increase of clearance. A number of phenomena associated with non-linear dynamics are observed, for instance: bistability, chaotic vibrations, windows of periodical vibrations, jump of amplitude, period-doubling cascade leading to chaos, bifurcation directly leading to chaos, and self-similarity of a Poincaré section. Moreover, amplitudes of vibrations are presented as functions of clearance. This provides an opportunity to select failure modes. Unfortunately, relations clearance - Amplitude and amplitude - clearance are ambiguous. Copyright © 2014 Trans Tech Publications Ltd, Switzerland. Source

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No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Bovine Pol II was prepared as described11 with modifications. Unless otherwise noted, all steps were completed at 4 °C. Protease inhibitors included 1 mM PMSF, 1 mM benzamidine, 60 μM leupeptin, and 200 μM pepstatin. Calf thymus was homogenized for 3 min in buffer A (50 mM Tris, pH 7.9 at 4 °C, 1 mM EDTA, 10 μM ZnCl , 10% glycerol, 1 mM DTT, protease inhibitors) using a 2 l blender (Waring). The homogenized material was centrifuged and the supernatant filtered through two layers of Miracloth. A 5% solution of polyethyleneimine, pH 7.9 at 25 °C, was added to a final concentration of 0.02%, and the material was stirred for 10 min then centrifuged. The resulting pellets were washed with buffer A before resuspension in buffer A (0.15 M ammonium sulfate). After centrifugation, the conductivity of the supernatant was adjusted to that of buffer A (0.2 M ammonium sulfate), and the resulting material was loaded on a 225-ml MacroPrepQ column equilibrated in buffer A (0.2 M ammonium sulfate). The column was washed with two column volumes of buffer A (0.2 M ammonium sulfate), followed by Pol II elution with buffer A (0.4 M ammonium sulfate). The eluate was precipitated by addition of finely ground ammonium sulfate added to 50% saturation, and pellets were collected by centrifugation. The pellets were resuspended in buffer A, and the conductivity was adjusted to that of buffer A (0.15 M ammonium sulfate). The material was clarified by centrifugation, and further purified using a 5-ml gravity flow column of 8WG16 (αRPB1 CTD) antibody-coupled sepharose equilibrated in buffer A (0.15 M ammonium sulfate). After application of the input material, the antibody column was washed with five column volumes of buffer A (0.5 M ammonium sulfate), sealed, and allowed to equilibrate to room temperature (20–25 °C) for 15 min. Pol II was eluted using buffer A (0.5 M ammonium sulfate, 50% (v/v) glycerol), and Pol-II-containing fractions were immediately mixed with buffer A (2 mM DTT, lacking glycerol and protease inhibitors). The diluted material was centrifuged and subjected to anion exchange chromatography using a UNO-Q column equilibrated in buffer A (0.1 M ammonium sulfate, 2 mM DTT, lacking protease inhibitors). Pol II was eluted using a linear gradient from 0.1 M to 0.5 M ammonium sulfate in buffer A (2 mM DTT, lacking protease inhibitors). For the purification of 12-subunit bovine Pol II, the Gdown1-free Pol II fraction was applied to a Sephacryl S-300 HiLoad sizing column equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). For the purification of bovine Pol II containing Gdown1, the Gdown1-free Pol II fraction was incubated with a 3× molar excess of human Gdown1 for 1 h at 4 °C before application to the Sephacryl S-300 HiLoad sizing column. Pol-II-containing fractions were concentrated using a 100-kDa cutoff Amicon concentrator to a final concentration of 2–4 mg ml−1. Gene-optimized human Gdown1 (Life Technologies) was cloned into pOPINB (N-terminal His tag and 3C protease site). After transformation, Escherichia coli BL21(DE3)RIL cells were grown at 37 °C in Lysogeny broth (LB) medium to an absorbance at 600 nm, A , of 0.5 before protein expression with 0.5 mM IPTG for 3–4 h at 37 °C. Subsequent steps were completed at 4 °C unless otherwise noted. Cells were lysed by sonication in buffer C (50 mM HEPES pH 7.5 (25 °C), 300 mM NaCl, 1 mM CaCl , 10% glycerol) supplemented with 10 mM imidazole, 1 mM PMSF, 1 mM benzamidine, 1 mM sodium metabisulfite, 1 mM DTT, and 2 μg ml−1 DNase I. Cleared lysate was subjected to affinity chromatography using Ni-NTA agarose (Qiagen), and excess chaperone was removed by washing the resin with a 5 mM ATP and 2 mg ml−1 denatured E. coli protein wash at room temperature in buffer C supplemented as above containing 30 mM imizdazole. Protein was eluted with buffer C supplemented as above, but lacking DNase I and containing 250 mM imidazole. Elutions were exchanged into buffer C supplemented with 10 mM imidazole and 1 mM DTT via a PD10 desalting column, followed by 3C protease cleavage at 4 °C overnight. Cleaved Gdown1 was subjected to reverse chromatography (Ni-NTA agarose) followed by dilution with buffer D (50 mM HEPES pH 7.5 (25 °C), 1 mM CaCl , 10% glycerol, 2 mM DTT) to a conductivity of buffer D containing 0.05 M NaCl. Diluted protein was subjected to cation exchange chromatography (MonoS 5/50) to remove additional chaperone, and eluted with a linear gradient from 0.05 M to 0.5 M NaCl in buffer D. The conductivity of the Gdown1-containing fractions was again adjusted to that of buffer D containing 0.05 M NaCl, and applied to a MonoQ 5/50 anion exchange column. Gdown1 was eluted using a linear gradient from 0.05 M to 0.5 M NaCl in buffer D. Fractions containing purified Gdown1 were pooled, resulting in a final concentration of 1–1.5 mg ml−1. Yield was approximately 2.5 mg per 2 l of E. coli culture. Purification of human SPT4 and SPT5 was as described31, with adaptations. Gene-optimized human SPT5 (pMK vector, no tag) and SPT4 were purchased from Life Technologies, and SPT4 was recloned into pOPINJ (N-terminal HIS6 and GST tags followed by a 3C protease cleavage site). SPT4 and SPT5 vectors were co-transformed into E. coli BL21(DE3)RIL cells, which were then grown at 37 °C in LB medium supplemented with 10 μM ZnCl to A  = 0.6. Expression was induced with 1 mM IPTG for 18 h at 18 °C. Cells were lysed by sonication in buffer E (25 mM Tris pH 7.4 (4 °C), 500 mM NaCl, 10 μM ZnCl , 5 mM DTT) supplemented with 5 mM imidazole and protease inhibitors (1 mM PMSF, 1 mM benzamidine, 60 μM leupeptin, and 200 μM pepstatin). Soluble material was passed over a Ni-NTA agarose column and washed with ten column volumes each of buffer E supplemented with 20 mM or 40 mM imidazole before elution in buffer E supplemented with 300 mM imidazole. Eluted protein was cleaved with 3C protease during overnight dialysis (4 °C) against buffer E, then subjected to reverse chromatography. Protein was passed over a HiTrap Q HP anion exchange column to remove DNA, and the flow-through fraction containing SPT4/5 was concentrated using a 50-kDa cutoff Amicon concentrator to 1–4 mg ml−1. The nucleic-acid scaffold (Metabion) was only slightly modified from what was used for a yeast Pol II EC crystal structure20 by mutation of the DNA templating base from C to A to generate a fully mismatched open bubble and by removing the upstream and downstream CC overhangs. A 1.5× molar excess of pre-annealed template DNA (sequence 5′-AAGCTCAAGTACTTAAGCCTGGTCATTACTAGTACTGCC-3′), non-template DNA (sequence 5′-GGCAGTACTAGTAAACTAGTATTGAAAGTACTTGAGCTT-3′), and RNA (sequence 5′-UAUAUGCAUAAAGACCAGGC-3′) were incubated with Pol II–Gdown1 at 4 °C for 10 min, then 20 °C for 15 min. To increase the randomness of Pol II EC particle orientations, the resulting complex (0.85 μM) was crosslinked with 3 mM BS3 (Thermo) for 30 min at 30 °C, then quenched with 50 mM ammonium bicarbonate. Crosslinked complex was applied to a Superdex 200 increase 10/300 GL column equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). The nucleic-acid-containing peak was concentrated to ~0.3 mg ml−1 as described above and used immediately for cryo-EM grid preparation. Four microlitres of sample were applied to glow-discharged Quantifoil R 3.5/1 holey carbon grids, which were then blotted and plunge-frozen in liquid ethane using a Vitrobot (FEI). Data were acquired using an FEI Titan Krios operated in energy-filtered transmission electron microscopy (EFTEM) mode at 300 kV equipped with a Gatan K2 Summit direct detector. Automated data collection was performed using the TOM toolbox32. Movie images were collected at a nominal magnification of ×37,000 (1.35 Å per pixel) in ‘super-resolution mode’ (0.675 Å per pixel) at a dose rate of about nine electrons per pixel per second. Two movies were acquired per hole, and each movie encompassed a total dose of ~43 electrons per square ångström over 8 s fractionated into 40 frames (0.2 s each). Defocus values ranged from −0.6 μm to −3.1 μm. Movies were aligned and binned as previously described15, 33, except that images were not partitioned into quadrants. Unless otherwise noted, processing was performed using RELION 1.3 (ref. 34). Contrast transfer function (CTF) parameters were estimated using CTFFIND4 (ref. 35). Initial 2D classes were generated after semi-automated picking of ~10,000 particles (box size 204) using e2boxer.py (EMAN2)36. Sixteen distinct classes were low-pass filtered to 25 Å resolution and used as templates for autopicking37, resulting in 476,100 particles selected from 1,172 micrographs. The autopicked particles were subjected to manual screening followed by screening by 2D classification, yielding an input data set of 409,401 particles. A previously published 22-Å-resolution cryo-negative stain reconstruction of human Pol II (EMD-1282)4 filtered to 50 Å was used as an initial reference for 3D refinement. Before any 3D classification, data were subjected to the particle polishing movie-processing algorithm of RELION 1.3 (ref. 38), resulting in an improvement in resolution from 3.7 Å to 3.4 Å. Three-dimensional classification was performed without image alignment as outlined in Extended Data Fig. 2. Masks were chosen to include either the entire Pol II EC or a smaller region of interest. The full data set was used as input for the classification of heterogeneity in the region of upstream DNA density. Only classes displaying strong clamp density were used as input for classification of conformations of the RPB4–RPB7 stalk. After classification, data were again subjected to 3D refinement with a 50 Å filtered reference volume. B-factors were automatically estimated in RELION39 and resolutions were reported on the basis of the gold-standard Fourier shell correlation (FSC) (0.143 criterion)40 as described41. The Pol II EC1 reconstruction was calculated from 264,134 particles to 3.4 Å resolution and sharpened with a B-factor of −137 Å2. The Pol II EC2 (improved RPB4–RPB7 stalk density) reconstruction was calculated from 219,265 particles to 3.6 Å and sharpened with a B-factor of −128 Å2. The Pol II EC3 (improved upstream DNA density) reconstruction was calculated from 184,122 particles to 3.7 Å and sharpened with a B-factor of −123 Å2. Focused refinements were achieved by continuing a refinement of the full data set from the iteration at which local searches began, but replacing the mask encompassing the entire Pol II EC density with a soft mask around the region of interest and allowing the refinement to continue to convergence. Local resolution was calculated using a sliding window method as described15, 42, except that a single pair of half maps was used per estimation and local resolution was not capped at the nominal value. Figures were generated using UCSF Chimera43. Alignments for each Pol II subunit were generated using the Homo sapiens, Bos taurus, Drosophila melanogaster, Schizosaccharomyces pombe, and S. cerevisiae sequences followed by alignment using Clustal Omega44. The Pol II crystal structure PDB 4BBS45 was chosen as a reference, as it displayed good stereochemistry and included the most complete model of the RPB2 protrusion domain. A starting model of ten-subunit Pol II was generated using the CCP4 (ref. 46) program chainsaw47 along with the alignment of the B. taurus and S. cerevisiae sequences. Conserved amino acids were retained, and non-conserved amino acids were pruned back to the gamma atom. The starting model was placed in the Pol II EC density by fitting in UCSF Chimera43, followed by fitting of rigid body groups in COOT48. Groups for rigid body refinement were chosen on the basis of observations from Pol II X-ray studies and visual inspection of the initial fit to the density. In regions of sufficient density quality, the model was manually adjusted to include complete side chains, and missing or divergent regions were built in COOT using B-factor sharpened maps. Multiple maps were used during model building, including the densities for the refinement using all data, Pol II EC1, Pol II EC2, Pol II EC3, and focused refinements. To generate a complete EC model, one molecule (chains A and B) of the crystal structure of human RPB4–RPB7 (PDB 2C35)49 was docked into the Pol II EC2 map. Regions near the ten-subunit core were adjusted manually to fit the density. Amino-acid side chains (previously stubbed) were added to the model if side chain density was visible. Ideal B-form DNA was manually fitted into the Pol II EC3 upstream DNA density. To improve backbone geometry, the EC model was subjected to PHENIX real space refinement (global minimization and ADP refinement) into one of the three unsharpened EC maps using Ramachandran, rotamer, and nucleic-acid restraints50. EC3 was used for refinement of the upstream DNA (chain N residues 1–13 and chain T residues 27–39). Because of the lower local resolution of the distal end of the upstream DNA, residues 1–10 of the non-template strand and residues 29–39 of the template strand were replaced with ideal B-form DNA that had been aligned to the refined upstream nucleic acids. EC2 was used for refinement of RPB4 and RPB7, and EC1 was used for the remaining model (EC body). The EC body was additionally refined as described above using the sharpened EC1 map to better position well-resolved side chains and nucleic acids within the density50. The final model was validated using Molprobity51, EMRinger52, and the FSC of the final model versus the EC1 map (Extended Data Fig. 3b). For model versus map FSC calculations, the EC1 map was masked using the RELION-generated soft automask used in postprocessing. Nucleic-acid scaffold used to assemble Pol II EC complexes was modified to include a 50 nucleotide RNA (sequence 5′-GAACGAGAUCAUAACAUUUGAACAAGAAUAUAUAUACAUAAAGACCAGGC-3′), as previous data have shown that DSIF affinity for ECs is increased as RNA length increases53. RNA was produced and purified as previously described54. A twofold molar excess of pre-annealed RNA and template DNA was incubated with Pol II for 20 min at 25 °C, followed by incubation with fourfold excess of non-template DNA for an additional 20 min at 25 °C. A fivefold molar excess of DSIF was incubated with the resulting Pol II EC for 20 min at 25 °C. Pol II–DSIF EC sample was applied to two consecutive Superdex 200 10/300 size-exclusion columns equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). Purified Pol II–DSIF EC was concentrated to ~0.5 mg ml−1 (~0.74 μM) and crosslinked with 3 mM BS3 (BS3-d0/d12, Creative Molecules) as described above. Crosslinked sample was again applied to two Superdex 200 10/300 columns, resulting in ~ 25 μg material. Sample was digested with trypsin, and analysed as previously described15, 55. Pol II–DSIF EC complexes were prepared as described above. Sample (~30 μg ml−1) was applied to glow-discharged 400 mesh copper grids coated with continuous carbon (Plano EM) for 1 min, washed on 500 μl water for 30 s, floated for 20 s on three consecutive 20-μl drops of 2% uranyl formate stain, and blotted to remove excess stain. Data were collected using an FEI Tecnai Spirit operated at 120 kV and a magnification of ×90,600. Micrographs were collected using a defocus range from −1.0 to −1.5 μm with an FEI Eagle CCD (charge-coupled device) camera binned 2× (image dimensions 2,048 pixels × 2,048 pixels) at a pixel size of 3.31 Å. Semi-automatic picking using e2boxer.py (EMAN2) yielded 11,531 particles from 120 micrographs. Data were subjected to 3D classification in RELION (eight classes, no CTF correction) using the cryo-negative stain reconstruction of human Pol II (EMD-1282)4 low-pass filtered to 60 Å as an initial reference. The two highest populated classes (comprising 85% of the data) were further classified into two classes each, for a total of four classes in which Pol II features beyond 60 Å were recognizable. Two classes did not have discernible additional density compared with Pol II (42% of data). A third class (28% of the data) displayed additional density near the RPB4–RPB7 stalk. A final class (15% of the data) showed additional density over the Pol II DNA binding cleft, as well as additional density near the RPB4–RPB7 stalk. Refinement of this subset of data (1,630 particles) resulted in a 3D reconstruction at 26 Å resolution, revealing extra density consistent with results from crosslinking coupled to mass spectrometry, previous DSIF–RNA crosslinking56, and the published interaction between SPT5 and the Pol II clamp coiled-coil motif26. Activity assays were performed as described57, with modifications. For reactions using fully complementary template and non-template DNA sequences, Pol II ECs were assembled stepwise beginning with either 12-subunit bovine Pol II or 12-subunit bovine Pol II in complex with human Gdown1, as indicated. Per reaction, Pol II was pre-assembled for 20 min at 28 °C with a 0.5 molar ratio of 5′ 6-FAM-labelled 20-nucleotide RNA annealed to template DNA, followed by incubation with a 1.0 molar ratio of fully complementary non-template DNA. The RNA and DNA sequences were the same as for the Pol II EC, except for an additional 46 nucleotides of downstream DNA. The template DNA sequence was 5′-ACAAATTACTGGGAAGTCGACTATGCAATACAGGCATCATTTGATCAAGCTCAAGTACTTAAGCCTGGTCATTACTAGTACTGCC-3′; the non-template DNA sequence was 5′-GGCAGTACTAGTAATGACCAGGCTTAAGTACTTGAGCTTGATCAAATGATGCCTGTATTGCATAGTCGACTTCCCAGTAATTTGT-3′, and RNA sequence was 5′-UAUAUGCAUAAAGACCAGGC-3′. Transcription was allowed to proceed for 10 min at 30 °C in the presence of 1–100 μM nucleoside triphosphates (NTPs) as indicated, and 0.2 pmol product per reaction was visualized on a 15% denaturing urea polyacrylamide gel. Transcription assays were also performed on the bubble scaffold used for structural studies. Pol II–EC complexes were prepared as described for cryo-EM, except that the samples were not crosslinked and the 20-nucleotide RNA used for assembly was 5′-labelled with 6-FAM. Assembled complex was incubated with 10–1,000 μM UTP at 30 °C for 10 min, allowing the extension of the RNA by two additional nucleotides. Product was visualized on a 20% denaturing urea polyacrylamide gel and imaged using a Typhoon FLA 9500.

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