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RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.

Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

The 21-subunit Mediator complex transduces regulatory information from enhancers to promoters, and performs an essential role in the initiation of transcription in all eukaryotes. Structural information on two-thirds of the complex has been limited to coarse subunit mapping onto 2-D images from electron micrographs. We have performed chemical cross-linking and mass spectrometry, and combined the results with information from X-ray crystallography, homology modeling, and cryo-electron microscopy by an integrative modeling approach to determine a 3-D model of the entire Mediator complex. The approach is validated by the use of X-ray crystal structures as internal controls and by consistency with previous results from electron microscopy and yeast two-hybrid screens. The model shows the locations and orientations of all Mediator subunits, as well as subunit interfaces and some secondary structural elements. Segments of 20–40 amino acid residues are placed with an average precision of 20 Å. The model reveals roles of individual subunits in the organization of the complex. Inside a cell, proteins are made from instructions encoded by DNA. To produce a particular protein, a section of DNA within a gene is copied into a molecule of messenger ribonucleic acid (or mRNA). This process is called transcription and is carried out by an enzyme known as RNA polymerase. Transcription begins in a region of DNA called a promoter, which is found at the start of the gene. RNA polymerase is brought to the DNA by many proteins, including the so-called Mediator complex. Mediator receives signals from within the cell and from the environment, processes the information, and instructs RNA polymerase whether to transcribe the gene or not. Mediator performs this important role in all organisms from yeast to humans, but it is not clear how it works. A crucial step towards the solution of this problem is to understand the three-dimensional structure of the complex. Previous research using a technique called ‘electron microscopy’ showed that Mediator is composed of three modules, referred to as Head, Middle and Tail. The images from electron microscopy were not sufficiently detailed to reveal the organization of the proteins within these modules. An open-source Integrative Modeling Platform (IMP for short) was recently developed to arrive at structural models of large protein complexes from a combination of experimental data and computer models. Now, Robinson, Trnka, Pellarin et al. have used this platform to study the Mediator complex. First, Robinson, Trnka, Pellarin et al. collected experimental data on the structure of the Mediator complex using two approaches called ‘chemical cross-linking’ and ‘mass spectrometry’. This data was combined with biochemical and structural information from previous studies to generate a three-dimensional model of the structure of the entire Mediator using IMP. The model is detailed enough to show the location and orientation of all the proteins in the complex. For example, a protein called Med17 connects the Head and Middle modules, while another subunit—known as Med14—spans the entire complex and makes extensive contacts with other proteins in all three modules.

The structure of the general transcription factor IIB (TFIIB) in a complex with RNA polymerase II reveals three features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the \"dock\" domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a \"finger\" domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein (TBP)-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. The trajectory of promoter DNA determined by the C-terminal domain of TFIIB traverses sites of interaction with TFIIE, TFIIF, and TFIIH, serving to define their roles in the transcription initiation process.

Two-stage exams-where students complete part one of an exam closed book and independently and part two is completed open book and independently (two-stage independent, or TS-I) or collaboratively (two-stage collaborative, or TS-C)-provide a means to include collaborative learning in summative assessments. Collaborative learning has been shown to have positive benefits, including increased student engagement and learning. To try to improve student learning, as measured by improvement in exam scores, two sections of introductory geology were taught using two-stage exams. It was hypothesized that class scores would be higher for semesters using two-stage exams-whether part two was TS-C or TS-I-than for semesters using traditional (T) exams. The median attendance rate was approximately 67% for all testing methods and was significantly greater when exams were TS-C (69%) rather than TS-I (53%). The class score was significantly greater during semesters when exams were TS-C (81%) but was not significantly different between T and TS-I semesters. To assess individual student learning over time, part one of the first exam and part one of the comprehensive final exam were compared. Across the F and D grade ranges, improvement on individual exam scores was significantly greater for the TS-C semester than for the TS-I and T semesters. Student learning, as measured by individual exam scores, improved due to the use of TS-C exams. The improvement in class scores due to the collaborative portion of two-stage exams was independent of increased attendance rates, greater for the lower-achieving students, and not observable if part two of the exam was completed as a take-home exam (TS-I).

General transcription factor TFIIH, previously described as a 10-subunit complex, is essential for transcription and DNA repair. An eleventh subunit now identified, termed Tfb6, exhibits 45% sequence similarity to human nuclear mRNA export factor 5. Tfb6 dissociates from TFIIH as a heterodimer with the Ssl2 subunit, a DNA helicase that drives promoter melting for the initiation of transcription. Tfb6 does not, however, dissociate Ssl2 from TFIIH in the context of a fully assembled transcription preinitiation complex. Our findings suggest a dynamic state of Ssl2, allowing its engagement in multiple cellular processes.

Sharing research equipment and personnel across investigators and laboratories has a long-standing history within research universities. However, the coordinated management of centralized, shared resources (i.e., core facilities) that provide access to instruments, technologies, services, expert consultation, and/or other scientific and clinical capabilities by Chief Research Officers (CROs) represents a more recent shift within the academy. While a number of recent surveys and studies have focused on the experiences of core facility directors and users, there has not yet been a targeted survey of CROs. Partnering with the Association for Public and Land Grant Universities Council on Research, fifty-eight CROs (or their designee) from research universities completed an electronic survey on core facilities (response rate = 35%). Core facilities formally reported to a range of entities within the university (and many to multiple entities), including the CRO office (83%), colleges/schools (67%), institutes/centers (42%), and departments (42%). Forty percent of respondents indicated that their university does not have a formal process to become and/or retain status as a recognized core facility. CROs also perceived that different types of core facilities directors differed in their general effectiveness (F(3,179)=6.88, p<.001); professional staff and administrators were rated as significantly more effective at directing/supervising core facilities than were tenure/tenure-track faculty (Tukey'spost-hoc;p<.005). Core facilities were funded through a variety of mechanisms, with the most common being use fees (96%), central and/or decentralized funding of directors or staff (77%), annual general fund allocation (62%), a designated portion of Facilities & Administration (F&A) reimbursements (46%), and internal grant programs (31%). Funds for purchasing new equipment within core facilities came from a number of sources, with the most common being external grants (87%), central institutional funds (83%), college/school/department funds (73%), use fees (50%), F&A resources (50%), and donations (27%). There are significant challenges to managing and funding core facilities; the present study provides new insights into the various strategies and tactics being taken by CROs to address these real and perceived challenges.

R.C. Davis provided the classic account of the European medieval world; equipping generations of undergraduate and ‘A’ level students with sufficient grasp of the period to debate diverse historical perspectives and reputations. His book has been important grounding for both modernists required to take a course in medieval history, and those who seek to specialise in the medieval period. In updating this classic work to a third edition, the additional author now enables students to see history in action ; the diverse viewpoints and important research that has been undertaken since Davis’ second edition, and progressed historical understanding. Each of Davis original chapters now concludes with a ‘new directions and developments’ section by Professor RI Moore, Emeritus of Newcastle University. A key work updated in a method that both enhances subject understanding and sets important research in its wider context. A vital resource, now up-to-date for generations of historians to come. Part One: The Dark Ages Introduction 1. Constantine the Great: The New Rome and Christianity 2. The barbarian invasions 3. Three reactions to the barbarian invasions 4. The Church and the Papacy 5. Islam 6. The Franks 7. The Break-up of the Carolingian Empire 8. Europe at the end of the ninth century: economic survey Part Two: The High Middle Ages (900-1250) Introduction. 1. The Saxon Empire 2. The Reform of the Papcy 3. Monasticism in the 11th and 12th centuries 4. Jerusalem regained and lost: the first three Crusades. 5. Feudal monachy and the French Kingdom (1066-1223) 6.The Emperor Frederrick I Barbarossa (1152-1190) 7. The Crisis of the Church 8. The new era in monachy 9. Europe in the middle of the 13th century: an economic survey Epilogue: the Mongols

Sharing research equipment and personnel across investigators and laboratories has a long-standing history within research universities. However, the coordinated management of centralized, shared resources (i.e., core facilities) that provide access to instruments, technologies, services, expert consultation, and/or other scientific and clinical capabilities by Chief Research Officers (CROs) represents a more recent shift within the academy. While a number of recent surveys and studies have focused on the experiences of core facility directors and users, there has not yet been a targeted survey of CROs. Partnering with the Association for Public and Land Grant Universities Council on Research, fifty-eight CROs (or their designee) from research universities completed an electronic survey on core facilities (response rate = 35%). Core facilities formally reported to a range of entities within the university (and many to multiple entities), including the CRO office (83%), colleges/schools (67%), institutes/centers (42%), and departments (42%). Forty percent of respondents indicated that their university does not have a formal process to become and/or retain status as a recognized core facility. CROs also perceived that different types of core facilities directors differed in their general effectiveness (F(3,179)=6.88, p<.001); professional staff and administrators were rated as significantly more effective at directing/supervising core facilities than were tenure/tenure-track faculty (Tukey's post-hoc; p<.005). Core facilities were funded through a variety of mechanisms, with the most common being use fees (96%), central and/or decentralized funding of directors or staff(77%), annual general fund allocation (62%), a designated portion of Facilities & Administration (F&A) reimbursements (46%), and internal grant programs (31%). Funds for purchasing new equipment within core facilities came from a number of sources, with the most common being external grants (87%), central institutional funds (83%), college/school/department funds (73%), use fees (50%), F&A resources (50%), and donations (27%). There are significant challenges to managing and funding core facilities; the present study provides new insights into the various strategies and tactics being taken by CROs to address these real and perceived challenges.