Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

Nogi, Yasuhisa; Yano, Ryoji; Nomura, Masayasu

doi:10.1073/pnas.88.9.3962

Cited by 165 publications

(166 citation statements)

References 34 publications

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“…Possibly any nuclear RNA with appropriate rRNA processing signals can be matured into 18S, 5.8S, and 25S rRNA. Indeed, it has been shown that rRNA transcribed from a GAL1 Pol II promoter construct is still able to produce functional rRNA (30). (28).…”

Section: Resultsmentioning

confidence: 99%

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

Prescott

Osheim

Jones

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor. Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae. These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains. However, deletion mutants for the 3 end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer. For ⌬rpa12, these spacer polymerases are devoid of nascent transcripts, suggesting they are immediately degraded. The homology of Rpa12p to the small subunit Rpb9p of Pol II and Rpc11p of Pol III, both implicated in transcriptional termination, points to a common termination mechanism for all three classes of RNA polymerase.

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Section: Resultsmentioning

confidence: 99%

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

Prescott

Osheim

Jones

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…When the intron sequences were cloned into a yeast plasmid expressing the full-length 35S pre-rRNA from the GAL7 promoter ( Fig. 1A; Nogi et al 1991), we again observed little unspliced pre-rRNA on Northern blots (Fig. 1B).…”

Section: In Vivo Splicing Is Linked To Rrnamentioning

confidence: 99%

“…Full-length 35S pre-rRNA was expressed from pNOY102, which contains the GAL7 promoter fused to a single copy of the 35S rRNA operon (6922 bp) from S. cerevisiae (Nogi et al 1991) and was the gift from M. Nomura. pNOY102-Tag was the gift of B. Peculis and contains a unique 18-nt sequence tag in the 59 end of the 25S rRNA (Peculis and Greer 1998).…”

Section: Plasmid Constructionmentioning

confidence: 99%

Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast

Jackson¹,

Koduvayur²,

Woodson³

2006

RNA

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Stable RNAs must form specific three-dimensional structures, yet many RNAs become kinetically trapped in misfolded conformations. To understand the factors that control the accuracy of RNA folding in the cell, the self-splicing activity of the Tetrahymena group I intron was compared in different genetic contexts in budding yeast. The extent of splicing was 98% when the intron was placed in its natural rDNA context, but only 3% when the intron was expressed in an exogenous pre-mRNA. Further experiments showed that the probability of forming the active intron structure depends on local sequence context and transcription by Pol I. Pre-rRNAs decayed at similar rates, whether the intron was wild type or inactivated by an internal deletion, suggesting that most of the unreacted pre-rRNA is incompetent to splice. Northern blots and complementation assays showed that mutations that destabilize the intron tertiary structure inhibited self-splicing and processing of internal transcribed spacer 2. The data are consistent with partitioning of pre-rRNAs into active and inactive populations. The misfolded RNAs are sequestered and degraded without refolding to a significant extent. Thus, the initial fidelity of folding can dictate the intracellular fate of transcripts containing this group I intron.

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“…Yeast A135 harboring mutation that abolishes A135 interaction with AC40/AC19 in two-hybrid system is functional. Yeast tester strain NOY308 -1a, which lacks A135 and is kept viable by the presence of Gal7-rDNA plasmid (26), was transformed with vector plasmid or plasmids expressing wild-type A135, or mutant A135 with D935A substitution. Transformants were spotted on rich medium containing galactose (top row) or glucose (bottom row) as carbon source.…”

Section: Discussionmentioning

confidence: 99%

“…To test the functional consequence of D935A substitution in A135, which abolishes the A135-AC19/AC40 interaction in the two hybrid assay, yeast plasmid expressing the mutant A135 gene was constructed. The phenotype of this gene was tested using haploid NOY408 -1a yeast tester strain (26). In this strain the chromosomal copy of A135 has been deleted, and the cells are kept viable because of the presence of a multicopy plasmid harboring an rDNA repeat under the control of GAL7 (RNAP II) promoter.…”

Section: Fig 2 a Strong Intrasubunit Interaction Between ␤ Segmentsmentioning

confidence: 99%

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

Naryshkina¹,

Rogulja²,

Golub³

et al. 2000

Journal of Biological Chemistry

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We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) ␣ and ␤ subunits during the formation of ␣ 2 ␤, a physiological RNAP assembly intermediate. We show that a 430-amino acidlong fragment containing ␤ conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with ␣. The ␣-interacting domain is held together by protein-protein interactions between ␤ segments F and I. Residues in catalytically important ␤ segments H and I directly participate in ␣ binding; substitutions of strictly conserved segment H Asp 1084 and segment I Gly 1215 abolish ␣ 2 ␤ formation in vitro and are lethal in vivo. The importance of these ␤ amino acids in ␣ binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of ␤ Asp 1084 in A135, the ␤-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest ␣-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli ␣-␤ interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.Cellular RNA polymerases (RNAPs) 1 are large, multisubunit enzymes. A typical prokaryotic RNAP core contains 5 polypeptides with a total molecular mass of ϳ400 kDa. Core RNAP from eukaryotes and archaea contain 10 -14 subunits with a total molecular mass in excess of 500 kDa. Sequence alignments of RNAP subunits reveal extensive similarities; each of the two largest RNAP subunits, which are the most evolutionarily conserved, contains 8 -9 colinear segments with many invariant amino acids (1, 2). RNAPs from different sources are also homologous structurally; low resolution (16 -35 Å) threedimensional models of Escherichia coli, and RNAP II and RNAP I from yeast obtained by means of electron crystallography reveal significant similarities (3-5).Evolutionarily conserved subunit segments probably form distinct functional domains common to all RNAPs. Genetic data support this notion; mutational changes of conserved residues selectively destroy distinct partial functions of the enzyme (e.g. the transition from abortive initiation to productive elongation (Ref. 6), or phosphodiester bond synthesis (Ref. 7)), but leave other functions unperturbed. However, isolated subunits themselves do not possess any of the partial functions of the whole enzyme (8). Therefore, RNAP functional sites are either formed allosterically upon the enzyme assembly, or are located at subunit interfaces. Thus, understanding inter-and intrasubunit interactions should provide insights in RNAP mechanism and regulation.E. coli RNAP assembles in vivo and in vitro according to the following scheme: ␣-␣ 2 -␣ 2 ␤-␣ 2 ␤␤Ј (9). The ␣ 2 ␤ assembly intermediate appears...

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Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

Cited by 165 publications

References 34 publications

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p

Self-splicing of a group I intron reveals partitioning of native and misfolded RNA populations in yeast

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

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