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Localization of the yeast RNA polymerase I-specific subunits.

ABSTRACT: The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.

SUBMITTER: Bischler N

PROVIDER: S-EPMC126139 | biostudies-literature | 2002 Aug

REPOSITORIES: biostudies-literature

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Localization of the yeast RNA polymerase I-specific subunits.

Bischler Nicolas N Brino Laurent L Carles Christophe C Riva Michel M Tschochner Herbert H Mallouh Véronique V Schultz Patrick P

The EMBO journal 20020801 15

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were cha ...[more]

PMID: 12145213

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Project description:All sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, ?1 and ?2. In all other bacteria studied to date, a dimer of identical ? subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition ?2??'). Based on an observation that both ?1 and ?2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell.By in vitro assembly from fully denatured state, we determined that both Francisella ? subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the ?1 and ?2 N-terminal domains (NTDs, responsible for dimerization). NTDs of ?2 did not interact detectably, while weak interaction between ?1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (?', ?) and ?1. No activity was observed with RNAP reconstitution reactions containing ?2, while robust transcription activity was detected in reactions containing ?1 and ?2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within ?-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.The results strongly suggest that most Francisella RNAP contains ? heterodimer with a minor subfraction possibly containing ?1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, ?1, interacts with the ? subunit during the ?2? RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.

Dataset Information

Localization of the yeast RNA polymerase I-specific subunits.

Publications

Localization of the yeast RNA polymerase I-specific subunits.

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