Specific features of RNA polymerases I and III:Structure and assembly

RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). In terms of subunit number and structure, RNAPI and RNAPIII are more complex than RNAPII that synthesizes thousands of different mRNAs. Specific subunits of the yeast RNAPI and RNAPIII form associated subcomplexes that are related to parts of the RNAPII initiation factors. Prior to their delivery to the nucleus where they function, RNAP complexes are assembled at least partially in the cytoplasm. Yeast RNAPI and RNAPIII share heterodimer Rpc40-Rpc19, a functional equivalent to the αα homodimer which initiates assembly of prokaryotic RNAP. In the process of yeast RNAPI and RNAPIII biogenesis, Rpc40 and Rpc19 form the assembly platform together with two small, bona fide eukaryotic subunits, Rpb10 and Rpb12. We propose that this assembly platform is co-translationally seeded while the Rpb10 subunit is synthesized by cytoplasmic ribosome machinery. The translation of Rpb10 is stimulated by Rbs1 protein, which binds to the 3′-untranslated region of RPB10 mRNA and hypothetically brings together Rpc19 and Rpc40 subunits to form the αα-like heterodimer. We suggest that such a co-translational mechanism is involved in the assembly of RNAPI and RNAPIII complexes

Characterization of the Lipopolysaccharide from a \u3cem\u3eRhizobium phaseoli\u3c/em\u3e Mutant that is Defective in Infection Thread Development

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A\u27s from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain

The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3' untranslated regions (3'UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism

sedFlow – a tool for simulating fractional bedload transport and longitudinal profile evolution in mountain streams

Especially in mountainous environments, the prediction of sediment dynamics is important for managing natural hazards, assessing in-stream habitats and understanding geomorphic evolution. We present the new modelling tool {sedFlow} for simulating fractional bedload transport dynamics in mountain streams. sedFlow is a one-dimensional model that aims to realistically reproduce the total transport volumes and overall morphodynamic changes resulting from sediment transport events such as major floods. The model is intended for temporal scales from the individual event (several hours to few days) up to longer-term evolution of stream channels (several years). The envisaged spatial scale covers complete catchments at a spatial discretisation of several tens of metres to a few hundreds of metres. sedFlow can deal with the effects of streambeds that slope uphill in a downstream direction and uses recently proposed and tested approaches for quantifying macro-roughness effects in steep channels. sedFlow offers different options for bedload transport equations, flow-resistance relationships and other elements which can be selected to fit the current application in a particular catchment. Local grain-size distributions are dynamically adjusted according to the transport dynamics of each grain-size fraction. sedFlow features fast calculations and straightforward pre- and postprocessing of simulation data. The high simulation speed allows for simulations of several years, which can be used, e.g., to assess the long-term impact of river engineering works or climate change effects. In combination with the straightforward pre- and postprocessing, the fast calculations facilitate efficient workflows for the simulation of individual flood events, because the modeller gets the immediate results as direct feedback to the selected parameter inputs. The model is provided together with its complete source code free of charge under the terms of the GNU General Public License (GPL) (www.wsl.ch/sedFlow). Examples of the application of sedFlow are given in a companion article by Heimann et al. (2015)

Reprogramming mRNA expression in response to defect in RNA polymerase III assembly in the yeast saccharomyces cerevisiae

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness

The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression

Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing