Structural basis of TFIIH activation for nucleotide excision repair.

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway

Structure of an inactive RNA polymerase II dimer

Eukaryotic gene transcription is carried out by three RNA polymerases: Pol I, Pol II and Pol III. Although it has long been known that Pol I can form homodimers, it is unclear whether and how the two other RNA polymerases dimerize. Here we present the cryo-electron microscopy (cryo-EM) structure of a mammalian Pol II dimer at 3.5 Å resolution. The structure differs from the Pol I dimer and reveals that one Pol II copy uses its RPB4-RPB7 stalk to penetrate the active centre cleft of the other copy, and vice versa, giving rise to a molecular handshake. The polymerase clamp domain is displaced and mobile, and the RPB7 oligonucleotide-binding fold mimics the DNA–RNA hybrid that occupies the cleft during active transcription. The Pol II dimer is incompatible with nucleic acid binding as required for transcription and may represent an inactive storage form of the polymerase

Structural basis of RNA processing by human mitochondrial RNase P

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria

Structure of the human Mediator-RNA polymerase II pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation at eukaryotic genes1–3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1–6. Here we prepare a recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and determine the structure of the complex by cryo-EM. Mediator uses its head module to contact the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7–9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the linker to the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate CTD phosphorylation

Cryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2

Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution. The structure shows that RPAP2 binds between the jaw domains of the polymerase subunits RPB1 and RPB5. RPAP2 is incompatible with binding of downstream DNA during transcription and is displaced upon formation of a transcription pre-initiation complex

Histone H1 binding to nucleosome arrays depends on linker DNA length and trajectory

Throughout the genome, nucleosomes often form regular arrays that differ in nucleosome repeat length (NRL), occupancy of linker histone H1 and transcriptional activity. Here, we report cryo-EM structures of human H1-containing tetranucleosome arrays with four physiologically relevant NRLs. The structures show a zig-zag arrangement of nucleosomes, with nucleosomes 1 and 3 forming a stack. H1 binding to stacked nucleosomes depends on the NRL, whereas H1 always binds to the non-stacked nucleosomes 2 and 4. Short NRLs lead to altered trajectories of linker DNA, and these altered trajectories sterically impair H1 binding to the stacked nucleosomes in our structures. As the NRL increases, linker DNA trajectories relax, enabling H1 contacts and binding. Our results provide an explanation for why arrays with short NRLs are depleted of H1 and suited for transcription, whereas arrays with long NRLs show full H1 occupancy and can form transcriptionally silent heterochromatin regions

Job satisfaction and career development of men in nursing

Background: Men are a growing minority in nursing. A deeper understanding of their motivation, career paths, and job satisfaction is needed to target recruitment and retention efforts. Currently the largest male cohort entering nursing is by second career. This study examines if job satisfaction differs for men who choose nursing as their first choice of career, as a convenience after exploring other options, or as a second career. Methods: This mixed-method study examined the relationship between men entering nursing by choice, convenience or second career and their resultant job satisfaction. A convenience sample of 238 men answered an online survey. Entry path, current practice position, and future career plans in five years was by self report. Job satisfaction was measured using Lester’s instrument. Results: Second career men were significantly more likely to work in specialty practice and more likely to aspire to advanced practice than those entering by choice or convenience early in their careers. No significant differences in job satisfaction were found between entry paths. Conclusions: These data raise provocative issues relating to recruiting men and the impact of men on the profession of nursing. Despite the steady but slow growth in the percent of men in nursing, its image remains overwhelmingly female. This survey does not identify substantial differences in job satisfaction between men who enter through different career paths or substantial differences with other studies of male and female nurses

Structure of replicating SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes1–3. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in active form, mimicking the replicating enzyme. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged ‘sliding poles’. These sliding poles can account for the known processivity of the RdRp that is required for replicating the long coronavirus genome3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19)4

Ion therapy within the trimodal management of superior sulcus tumors: the INKA trial

Background: The standard trimodal treatment concept in locally advanced and non-metastasized non-small-cell superior sulcus tumors consists of a preoperative chemoradiation followed by surgical resection. High linear energy transfer (LET) radiation as, for example, C12 heavy-ion beam therapy theoretically offers biological advantages compared to high energy x-ray therapy as, for example, higher biological efficiency. Methods/Design: In the present prospective, single-armed, open pilot study performed at the Heidelberg Ion-Beam Therapy Center (HIT) in Heidelberg, the radiation treatment within the standard trimodal concept will be exchanged against C12 heavy-ion beam treatment and apply 39GyE in 13 single fractions in combination with a chemotherapy consisting of cisplatin and vinorelbine (local standard). The primary endpoint is feasibility and safety measured by the incidence of NCI-CTCAE grade 3/4 toxicity and/or discontinuation due to any reason. Secondary endpoint is the degree of regression in the histological specimen. The main inclusion criteria are histologically confirmed non-small-cell superior sulcus tumor, nodal disease stage ≤ N2, Karnofsky performance score ≥70%, patient age between 18 and 75 years as well as written informed consent. The main exclusion criteria include medical contraindications against elements of the trimodal treatment concept, PET confirmed nodal disease stage N3, stage IV disease, prior thoracic irradiation and decompensated diseases of the lung, cardio-vascular system, metabolism, hematopoietic and coagulation system and renal function. Furthermore, patients with implanted active medical devices without certification for ion-beam therapy are not allowed to take part in the study. Trial registration number: DRKS00006323 (www.drks.de)

The structure of a dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs