SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus.

Abstract

SPT6 is an essential gene of Saccharomyces cerevisiae that appears to play a role in transcription. Mutations in the SPT6 (SSN20, CRE2) gene suppress 8 insertion mutations in the 5' regions of HIS4 and LYS2 and mutations in cis-and/or trans-acting elements that are required for expression of SUC2 and ADH2. We report here that SPT6 encodes a 170-kilodalton highly charged protein with an extremely acidic amino terminus. By use of an epitope-tagged SPT6 protein, we have determined by indirect immunofluorescence that the SPIT6 protein is located in the nucleus.Mutations in the Saccharomyces cerevisiae gene SPT6 (also called SSN20 [25,26] and CRE2 [7,8]) have been isolated as suppressors of both cis-and trans-acting mutations that alter transcription of particular genes. Mutations in SPT6 were first isolated as suppressors of an insertion mutation at the HIS4 gene (his4-9128) caused by a long terminal repeat of the transposable element Ty (32). Dominant and recessive mutations in SPT6 (called SSN20) were also isolated as extragenic suppressors of the defect in SUC2 expression caused by snf2 or snJ5 mutations and were shown to act as suppressors of snf6 (26) and SUC2 mutant alleles with upstream activation sequence deletions (25). One spt6 mutation, called cre2-1, was isolated in a selection for mutations that allow ADH2 to escape glucose repression. The cre2-1 mutation also restores a moderate level of ADH2 expression in the absence of the trans-acting positive activator ADRI (7,8). In every case that has been examined, suppression by spt6 mutations is mediated at the level of transcription (6, 25).The SPT6 gene was cloned by complementation of both an spt6 and an ssn20 allele, and a 4.6-kiobase (kb) mRNA was identified (6, 25). The gene was mapped to the right arm of chromosome VII (6,25). Null alleles of SPT6 were constructed and found to be lethal in haploid cells (6,25), consistent with the temperature-sensitive lethality conferred by many spt6 alleles (7,26,32). Experiments with the cloned SPT6 gene demonstrated that either increased or decreased dosage of the wild-type SPT6 gene also suppresses his4-9128 mutations (6) and SUC2 upstream regulatory region deletions (25). These results suggested that the SPT6 protein functions as part of a complex which is affected by altered stoichiometry (6,25). Since SPT6 appears to play an essential and general role in transcription, we have characterized further the gene and its product.Sequence of the SPT6 gene. The nucleotide sequence of the SPT6 gene was determined by the method of Sanger et al. (30). The open reading frame (Fig. 1) highly charged 1,451-amino-acid protein of 168,290 daltons (Da). In the entire protein, 19%o of the residues were acidic and 15% were basic. A striking characteristic of the predicted protein was its acidic amino terminus: the first 70 residues had a net charge of -30 (50%o acidic residues), and the amino-terminal third of the protein (484 residues) had a net charge of -81 (29% acidic residues). In addition, the SPT6 sequence included several...

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus.

“…Malone et al (32) have demonstrated that both overexpression (by increased copy number) and mutation of the CDC681SPT16 gene result in suppression of 8-element insertion mutations. In this respect the CDC681SPT16 gene is similar to the SPT5 (60) and SPT61SSN201CRE2 genes (6,59). Both SPT5 and SPT6 encode essential nuclear proteins with extremely acidic N termini and have been suggested to influence gene expression through a function in chromatin assembly or modification.…”

Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure.

“…Finally, histone octamers, but not H3-H4 tetramers, efficiently repress 5S RNA transcription in vitro (Tremethick et al 1990;Almouzni et al 1991). This model also raises the possibility that proteins functionally related to histones, such as the acidic SPT5 and SPT6 proteins (Swanson et al 1990, could repress transcription by stabilizing the interaction of H2A-H2B dimers with DNA-bound H3-H4 tetramers, in a manner analogous to that of the acidic chromatin assembly factor nucleoplasmin (Dingwall et al 1987). Other proteins implicated in affecting chromatin structure, including SIN1/SPT2, SPT4, and SPT16 Malone et al 1991;Swanson and Winston 1992), might also affect histone-histone or histone-DNA interactions.…”

A Tandem SH2 Domain in Transcription Elongation Factor Spt6 Binds the Phosphorylated RNA Polymerase II C-terminal Repeat Domain (CTD)

“…The C-terminal region of mouse Spt6 binds the Ser-2-phosphorylated CTD, 2 suggesting a mechanism for how Spt6 is recruited to elongating RNA polymerase II (12). The Spt6 C-terminal region was predicted to contain an SH2 domain.…”

“…Spt6 is an essential nuclear protein (1,2) that is required for transcription elongation together with Spt4 and Spt5 (3,4). Spt6 co-localizes with Spt5 on transcribed genes in Drosophila polytene chromosomes (5,6).…”