Co-expression of multiple subunits enables recombinant SNAPC assembly and function for transcription by human RNA polymerases II and III.
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.
Hanzlowsky
Andrej
A
Department of Chemistry, Michigan State University, East Lansing, USA.
Jelencic
Blanka
B
Jawdekar
Gauri
G
Hinkley
Craig S
CS
Geiger
James H
JH
Henry
R William
RW
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D021122:Protein Subunits; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; C106703:SNAPC protein complex, human; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D012319:RNA Polymerase II; D012320:RNA Polymerase III