The Reb1-homologue Ydr026c/Nsi1 is required for efficient RNA polymerase I termination in yeast.

Several DNA cis-elements and trans-acting factors were described to be involved in transcription termination and to release the elongating RNA polymerases from their templates. Different models for the molecular mechanism of transcription termination have been suggested for eukaryotic RNA polymerase I (Pol I) from results of in vitro and in vivo experiments. To analyse the molecular requirements for yeast RNA Pol I termination, an in vivo approach was used in which efficient termination resulted in growth inhibition. This led to the identification of a Myb-like protein, Ydr026c, as bona fide termination factor, now designated Nsi1 (NTS1 silencing protein 1), since it was very recently described as silencing factor of ribosomal DNA. Possible Nsi1 functions in regard to the mechanism of transcription termination are discussed.

TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production.

Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome-dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I-Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I-Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I-Rrn3p levels.

Site specific phosphorylation of yeast RNA polymerase I.

All nuclear RNA polymerases are phosphoprotein complexes. Yeast RNA polymerase I (Pol I) contains approximately 15 phosphate groups, distributed to 5 of the 14 subunits. Information about the function of the single phosphosites and their position in the primary, secondary and tertiary structure is lacking. We used a rapid and efficient way to purify yeast RNA Pol I to determine 13 phosphoserines and -threonines. Seven of these phosphoresidues could be located in the 3D-homology model for Pol I, five of them are more at the surface. The single phosphorylated residues were systematically mutated and the resulting strains and Pol I preparations were analyzed in cellular growth, Pol I composition, stability and genetic interaction with non-essential components of the transcription machinery. Surprisingly, all Pol I phosphorylations analyzed were found to be non-essential post-translational modifications. However, one mutation (subunit A190 S685D) led to higher growth rates in the presence of 6AU or under environmental stress conditions, and was synthetically lethal with a deletion of the Pol I subunit A12.2, suggesting a role in RNA cleavage/elongation or termination. Our results suggest that individual major or constitutively phosphorylated residues contribute to non-essential Pol I-functions.

Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity.

BACKGROUND: Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. RESULTS: Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity. CONCLUSIONS: The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Binding of the termination factor Nsi1 to its cognate DNA site is sufficient to terminate RNA polymerase I transcription in vitro and to induce termination in vivo.

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3' end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination.

Reduction in ribosomal protein synthesis is sufficient to explain major effects on ribosome production after short-term TOR inactivation in Saccharomyces cerevisiae.

Ribosome synthesis depends on nutrient availability, sensed by the target of rapamycin (TOR) signaling pathway in eukaryotes. TOR inactivation affects ribosome biogenesis at the level of rRNA gene transcription, expression of ribosomal proteins (r-proteins) and biogenesis factors, preribosome processing, and transport. Here, we demonstrate that upon TOR inactivation, levels of newly synthesized ribosomal subunits drop drastically before the integrity of the RNA polymerase I apparatus is severely impaired but in good correlation with a sharp decrease in r-protein production. Inhibition of translation by cycloheximide mimics the rRNA maturation defect observed immediately after TOR inactivation. Both cycloheximide addition and the depletion of individual r-proteins also reproduce TOR-dependent nucleolar entrapment of specific ribosomal precursor complexes. We suggest that shortage of newly synthesized r-proteins after short-term TOR inactivation is sufficient to explain most of the observed effects on ribosome production.