Molecular basis of RNA polymerase III transcription repression by Maf1

RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex. Binding of DNA and RNA causes ordering of the Pol III-specific subcomplex C82/34/31 that is required for transcription initiation. Maf1 binds the Pol III clamp and rearranges C82/34/31 at the rim of the active center cleft. This impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors Brf1 and TBP and thus prevents closed complex formation. Maf1 does however not impair binding of a DNA-RNA scaffold and RNA synthesis. These results explain how Maf1 specifically represses transcription initiation from Pol III promoters and indicate that Maf1 also prevents reinitiation by binding Pol III during transcription elongation

Conductive nitrides: growth principles, optical and electronic properties, and their perspectives in photonics and plasmonics

The nitrides of most of the group IVb-Vb-VIb transition metals (TiN, ZrN, HfN, VN, NbN, TaN, MoN, WN) constitute the unique category of conductive ceramics. Having substantial electronic conductivity, exceptionally high melting points and covering a wide range of work function values, they were considered for a variety of electronic applications, which include diffusion barriers in metallizations of integrated circuits, Ohmic contacts on compound semiconductors, and thin film resistors, since early eighties. Among them, TiN and ZrN are recently emerging as significant candidates for plasmonic applications. So the possible plasmonic activity of the rest of transition metal nitrides (TMN) emerges as an important open question. In this work, we exhaustively review the experimental and computational (mostly ab initio) works in the literature dealing with the optical properties and electronic structure of TMN spanning over three decades of time and employing all the available growth techniques. We critically evaluate the optical properties of all TMN and we model their predicted plasmonic response. Hence, we provide a solid understanding of the intrinsic (e.g. the valence electron configuration of the constituent metal) and extrinsic (e.g. point defects and microstructure) factors that dictate the plasmonic performance. Based on the reported optical spectra, we evaluate the quality factors for surface plasmon polariton and localized surface plasmon for various TMN and critically compare them to each other. We demonstrate that, indeed TiN and ZrN along with HfN are the most well-performing plasmonic materials in the visible range, while VN and NbN may be viable alternatives for plasmonic devices in the blue, violet and near UV ranges, albeit in expense of increased electronic loss. Furthermore, we consider the alloyed ternary TMN and by critical evaluation and comparison of the reported experimental and computational works, we identify the emerging optimal tunable plasmonic conductors among the immense number of alloying combinations

Self-assembled plasmonic templates produced by microwave annealing: applications to surface-enhanced Raman scattering

Perhaps the simplest method for creating metal nanoparticles on a substrate is by driving their self-assembly with the thermal annealing of a thin metal film. By properly tuning the annealing parameters one hopes to discover a recipe that allows the pre-determined design of the NP arrangement. However, thermal treatment is known for detrimental effects and is not really the manufacturer's route of choice when it comes to large-scale applications. An alternative method is the use of microwave annealing, a method that has never been applied for metal processing, due to the high reflectance of microwave radiation at the surface of a metal. However, in this work we challenge the widely used nanostructuring methods by proving the microwave's annealing ability to produce plasmonic templates, out of extremely thin metal films, by simply using a domestic microwave oven apparatus. We show that this process is generic and independent of the deposition method used for the metal and we further quantify the suitability of these plasmonic templates for use in surface-enhanced Raman scattering applications

Sensory Attenuation Assessed by Sensory Evoked Potentials in Functional Movement Disorders.

BACKGROUND: Functional (psychogenic) movement disorders (FMD) have features associated with voluntary movement (e.g. distractibility) but patients report movements to be out of their control. One explanation for this phenomenon is that sense of agency for movement is impaired. The phenomenon of reduction in the intensity of sensory experience when movement is self-generated and a reduction in sensory evoked potentials (SEPs) amplitude at the onset of self-paced movement (sensory attenuation) have been linked to sense of agency for movement. METHODS: We compared amplitude of SEPs from median nerve stimulation at rest and at the onset of a self-paced movement of the thumb in 17 patients with FMD and 17 healthy controls. RESULTS: Patients showed lack of attenuation of SEPs at the onset of movement compared to reduction in amplitude of SEPs in controls. FMD patients had significantly different ratios of movement onset to rest SEPs than did healthy controls at each electrode: 0.79 in healthy controls and 1.35 in patients at F3 (t = -4.22, p<0.001), 0.78 in healthy controls and 1.12 at patients C3 (t = -3.15, p = 0.004) and 0.77 in healthy controls and 1.05 at patients P3 (t = -2.88, p = 0.007). CONCLUSIONS: Patients with FMD have reduced sensory attenuation as measured by SEPs at onset of self-paced movement. This finding can be plausibly linked to impairment of sense of agency for movement in these patients

Structural basis of RNA polymerase III transcription initiation.

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4-4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation

DNA origami-based single-molecule forcespectroscopy elucidates RNA Polymerase IIIpre-initiation complex stability

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III

Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I

Background: Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I. Methodology/Principal Findings: By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription

Function and Assembly of a Chromatin-Associated RNase P that Is Required for Efficient Transcription by RNA Polymerase I

Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I.By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription.Our findings reveal that RNase P activates transcription of rDNA by Pol I through a novel assembly process and that this catalytic ribonucleoprotein determines the transcription output of Pol I and Pol III, two functionally coordinated transcription machineries

An RNA Polymerase III-Dependent Heterochromatin Barrier at Fission Yeast Centromere 1

Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNAAlanine gene barrier (cen1 tDNAAla) blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1) in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNAAla gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere