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

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. KeywordsTranscription; SNAP C ; Human snRNA; Co-expression; Escherichia coliIn eukaryotic organisms, transcription occurs by three different RNA polymerases I, II, and III that are unable to directly recognize promoter elements but instead require distinct assemblies of general transcription factors for efficient promoter recruitment. The initial step of promoter recognition by the general transcription machinery is a key step in transcription and is frequently targeted for intervention during gene regulation [1]. Interestingly, those components of the general transcription machinery that function directly in core promoter DNA binding Typically, promoter recognition complexes are also specialized for transcription by a single class of polymerase; however, the SNAP C general transcription factor [6], also known as PTF [7], provides an interesting example of functional versatility through its role in snRNA gene transcription by both RNA polymerases II and III. Regardless of polymerase specificity, the promoters of human snRNA genes are very similar containing a proximal sequence element (PSE) 1 within the core promoter region [reviewed in 8,9]. SNAP C binds to the PSE and depending upon the presence or absence of an adjacent TATA box participates in different pathways of pre-initiation complex assembly. The presence of a TATA box, such as in U6 snRNA genes, dictates a role for SNAP C in pre-initiation complex assembly for RNA polymerase III transcription along with TBP, as a component of TFIIIB [10,11]. In contrast, the absence of a TATA box, such ...

Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase

“…7C, D, and E), suggesting that SNAPc may be removed piecewise during promoter disassembly. Alternatively, the fragile state may render SNAPc 190 inaccessible to our antibody, an interpretation that is far more easily reconciled with previous observations that SNAPc 190 interacts with all the other SNAPc subunits (41) and that U2 transcription can be activated by a minimal complex of SNAPc 43,50,and 190 in which only SNAPc 50 and 190 can be UV cross-linked to the DNA (34).…”

“…We speculate below, based on this and other experiments, that the open state may facilitate polymerase engagement, initiation, and elongation by circumventing the TFIIH-dependent promoter melting and clearance steps (34,35,41,42). Curiously, only weak DNase I sensitivity is seen over the transcription initiation site on the template strand ( Fig.…”

“…7E]), Pol II occupancy is more complex: In CSB null cells with a mitotic index of Ͼ90%, the Pol II C-terminal domain (CTD) and serine-5P signals appear undiminished, although SNAPc 190 is low or lost (Fig. 7C); since SNAPc 190 is thought to form the backbone of the SNAPc complex and is required for initiation in vitro (34), this is consistent with our earlier hypothesis (111) that CSB may play a role in removing Pol II from the U2 transcription unit as metaphase approaches. Transduction with TAT-p53CTD has the same effect on SNAPc 43 and 190 as loss of CSB function, and the CTD and serine-5P signals also remain high (Fig.…”

“…We also found by ChIP that the SNAPc complex which binds the TATA-like PSE is removed at metaphase but remains bound under conditions that induce locusspecific metaphase fragility of the U2 genes, such as a loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric C terminus of p53. We propose that the U2 snRNA promoter establishes a persistently open state in which TFIIH is required for capping (72) but not for the potentially slow promoter melting step (34,35,41,42,48); this open transcriptional state would facilitate rapid initiation during interphase but require disassembly during metaphase to allow proper chromatin condensation.…”

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

“…Another concern is that some interacting proteins only became soluble when all partners were presented together (Kummel et al, 2005;Tundup et al, 2006). New methods based on co-expression strategy offered alternative way to facilitate the process of protein expression and protein-protein interaction, by using compatible vectors (Huppa and Ploegh, 1997;Fabian et al, 1998;Hanzlowsky et al, 2006), multiple promoters (Belyaev and Roy, 1993;Scheich et al, 2007), polycistrons (Selleck et al, 2005;Neumann et al, 2007;Bieniossek et al, 2009), fusion linkers (Clements et al, 2000) in both prokaryotic and eukaryotic systems. One advantageous application was that TEV (Tobacco Etch Virus) NIa protease carried out site-specific cleavage on polyprotein to yield multiple native proteins within E. coli using a single promoter (Shih et al, 2005;Chen et al, 2010).…”