The Unorthodox SNAP50 Zinc Finger Domain Contributes to Cooperative Promoter Recognition by Human SNAPC

Abstract

C through its zinc finger domain. The SNAP50 zinc finger domain contains 15 cysteine and histidine residues configured in two potential zinc coordination arrangements. Individual alanine substitution of each cysteine and histidine residue demonstrated that eight sites are important for DNA binding by SNAP C . However, metal binding studies revealed that SNAP C contains a single zinc atom indicating that only one coordination site functions as a zinc finger. Of the eight residues critical for DNA binding, four cysteine residues were also essential for both U1 and U6 transcription by RNA polymerase II and III, respectively. Surprisingly, the remaining four residues, although critical for U1 transcription could support partial U6 transcription. DNA binding studies showed that defects in DNA binding by SNAP C alone could be suppressed through cooperative DNA binding with another member of the RNA polymerase III general transcription machinery, TFIIIB. These results suggest that these eight cysteine and histidine residues perform different functions during DNA binding with those residues involved in zinc coordination likely performing a dominant role in domain stabilization and the others involved in DNA binding. These data further define the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding domain.The human snRNA 3 gene family is unusual in that related member genes are transcribed by either RNA polymerase II or III depending upon their core promoter structures, and they thus serve as an interesting model to understand principles of polymerase selection and activity both during normal and deregulated growth (reviewed in Refs. 1 and 2). Human snRNA genes are defined by the presence of a diagnostic promoter element called the proximal sequence element (PSE). For both polymerase systems, the PSE recruits the general transcription factor called SNAP C (3), which is also known as PTF (4).SNAP C is composed of at least five subunits SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19 (5-9). SNAP190 can interact with all the other subunits, and provides a central architectural backbone to the complex (10). SNAP190 directly interacts with the transcriptional activator protein Oct-1 during stimulated transcription of human snRNA genes (11, 12). In the RNA polymerase III pathway, SNAP190 also interacts with TBP to recruit Brf2-TFIIIB to the TATA box of human U6 snRNA genes (13,14). Thus, the upstream signal from Oct-1 is conveyed through SNAP190 stimulating preinitiation complex assembly with other general transcription factors as a prerequisite for RNA polymerase III recruitment. Interestingly, protein kinase CK2 can phosphorylate SNAP190 to impede DNA binding; however, promoter recognition by SNAP C can be restored by cooperative binding of TBP to those promoter sequences containing both a PSE and TATA box, but not sequences lacking the TATA element (15). This last observation suggests that CK2 can differentially influence RNA polymerase II and III snRNA transcription by covalent modification of SNAP C...

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

“…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.…”

“…DNase I-hypersensitive sites are regions of DNA where chromatin structure (or the lack of it) allows nuclease access. We did not pursue hypersensitive sites 3 and 2 because site 3 is located considerably upstream of the essential U2 snRNA promoter (2,41,42) and site 2 lies within the promoter, which is known to be highly structured (8).…”

“…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.…”

“…Intriguingly, recognition of the 3Ј end formation signal requires a U snRNA promoter (20, 36); mRNA promoters cannot substitute, suggesting that U snRNA promoters either prevent loading of mRNA-specific factors, such as the CPSF component of the polyadenylation apparatus, or facilitate loading of snRNAspecific factors, such as Integrator, both of which bind to the Pol II CTD (15, 24). Curiously, although U1 to U5 are transcribed by Pol II, the presence of a TATA box just downstream of the U6 PSE switches the promoter specificity from Pol II to Pol III; accordingly, U6 genes have a Pol III oligo(dT) termination signal instead of a Pol II 3Ј-end formation signal (35,41,42).In the primate lineage, where the abundance of U1 and U2 snRNA approaches that of the rRNAs, the genes encoding U1 and U2 are tandemly repeated. The human RNU1 locus spans 1.35 Mbp and contains about 30 tandemly repeated U1 snRNA genes; the individual repeat units are Ͼ45 kb in size and contain a single U1 snRNA gene interspersed with numerous tRNA genes (6, 101).…”

“…The genes encoding these U snRNAs are single copy in budding yeast, where introns are rare and U snRNPs are scarce, but in mammals, where almost all mRNAs have multiple introns, the major spliceosomal U snRNAs are encoded by multigene families and the U snRNPs are nearly as abundant as rRNA. U snRNA genes have been characterized for many species, and the major transcriptional signals and trans-acting factors are well defined, especially in mammals (35,41,42). U1 to U5 are transcribed by RNA polymerase II (Pol II).…”