A Map of Protein-Protein Contacts within the Small Nuclear RNA-activating Protein Complex SNAPc

Beicong Ma,Nouria Hernandez

doi:10.1074/jbc.m009301200

Abstract

The nucleation of RNA polymerases I-III transcription complexes is usually directed by distinct multisubunit factors. In the case of the human RNA polymerase II and III small nuclear RNA (snRNA) genes, whose core promoters consist of a proximal sequence element (PSE) and a PSE combined with a TATA box, respectively, the same multisubunit complex is involved in the establishment of RNA polymerase II and III initiation complexes. This factor, the snRNA-activating protein complex or SNAP(c), binds to the PSE of both types of promoters and contains five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. SNAP(c) binds cooperatively with both Oct-1, an activator of snRNA promoters, and in the RNA polymerase III snRNA promoters, with TATA-binding protein, which binds to the TATA box located downstream of the PSE. Here we have defined subunit domains required for SNAP(c) subunit-subunit association, and we show that complexes containing little more than the domains mapped here as required for subunit-subunit contacts bind specifically to the PSE. These data provide a detailed map of the subunit-subunit interactions within a multifunctional basal transcription complex.

Highlights

The basal transcription machineries that recruit RNA polymerases I–III to promoters are all composed of large multisubunit complexes
In the case of the human RNA polymerase II and III small nuclear RNA genes, whose core promoters consist of a proximal sequence element (PSE) and a PSE combined with a TATA box, respectively, the same multisubunit complex is involved in the establishment of RNA polymerase II and III initiation complexes
The core RNA polymerase II small nuclear RNA (snRNA) promoters consist of a single essential element, the proximal sequence element (PSE), whereas the core RNA polymerase III snRNA promoters consist of both a PSE and a TATA box

Summary

EXPERIMENTAL PROCEDURES

Translations in Vitro—The various truncated open reading frames were subcloned into derivatives of the expression vector pCite (Novagen). The pCite constructs were used as templates for in vitro transcription and translation with the TNT T7 coupled reticulocyte lysate system (Promega L4610). Two ␮g of DNA template were mixed with 25 ␮l of TNT rabbit reticulocyte lysate and (i) 2 ␮l of reaction buffer, (ii) 1 ␮l of 1 mM amino acids minus methionine mix, and (iii) 1 ␮l of 40 units/␮l RNasin ribonuclease inhibitor, all supplied by the manufacturer, and 2 ␮l of [35S]methionine at 10 mCi/ml in a total volume of 50 ␮l, and incubated for 30 min at 30 °C. The various subcomplexes were analyzed for DNA binding in an electromobility shift assay with a probe containing a high affinity mouse U6 PSE or a mutated PSE as described by Mittal and Hernandez (14)

RESULTS

DISCUSSION

Beicong Ma and Nouria Hernandez