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Protein Expr Purif. 2006 Aug;48(2):215-23. Epub 2006 Mar 15.

Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III.

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  • 1Department of Chemistry, Michigan State University, East Lansing, USA.

Abstract

Human small nuclear (sn) RNA genes are transcribed by either RNA polymerase II or III depending upon the arrangement of their core promoter elements. Regardless of polymerase specificity, these genes share a requirement for a general transcription factor called the snRNA activating protein complex or SNAP(C). This multi-subunit complex recognizes the proximal sequence element (PSE) commonly found in the upstream promoters of human snRNA genes. SNAP(C) consists of five subunits: SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. Previous studies have shown that a partial SNAP(C) composed of SNAP190 (1-514), SNAP50, and SNAP43 expressed in baculovirus is capable of PSE-specific DNA binding and transcription of human snRNA genes by RNA polymerases II and III. Expression in a baculovirus system yields active complex but the concentration of such material is insufficient for many bio-analytical methods. Herein, we describe the co-expression in Escherichia coli of a partial SNAP(C) containing SNAP190 (1-505), SNAP50, SNAP43, and SNAP19. The co-expressed complex binds DNA specifically and recruits TBP to U6 promoter DNA. Importantly, this partial complex functions in reconstituted transcription of both human U1 and U6 snRNA genes by RNA polymerases II and III, respectively. This co-expression system will facilitate the functional characterization of this unusual multi-protein transcription factor that plays an important early role for transcription by two different polymerases.

PMID:
16603380
[PubMed - indexed for MEDLINE]
PMCID:
PMC2714255
Free PMC Article
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