C-terminal processing of yeast Spt7 occurs in the absence of functional SAGA complex.

BACKGROUND: Spt7 is an integral component of the multi-subunit SAGA complex that is required for the expression of approximately 10% of yeast genes. Two forms of Spt7 have been identified, the second of which is truncated at its C-terminus and found in the SAGA-like (SLIK) complex. RESULTS: We have found that C-terminal processing of Spt7 to its SLIK form (Spt7SLIK) and to a distinct third form (Spt7Form3) occurs in the absence of the SAGA complex components Gcn5, Spt8, Ada1 and Spt20, the latter two of which are required for the integrity of the complex. In addition, N-terminally truncated derivatives of Spt7, including a derivative lacking the histone fold, are processed, indicating that the C-terminus of Spt7 is sufficient for processing and that processing does not require functional Spt7. Using galactose inducible Spt7 expression, we show that the three forms of Spt7 appear and disappear at approximately the same rate with full-length Spt7 not being chased into Spt7SLIK or Spt7Form3. Interestingly, reduced levels of Spt7SLIK and Spt7Form3 were observed in a strain lacking the SAGA component Ubp8, suggesting a regulatory role for Ubp8 in the truncation of Spt7. CONCLUSION: We conclude that truncation of Spt7 occurs early in the biosynthesis of distinct Spt7 containing complexes rather than being a dynamic process linked to the action of the SAGA complex in transcriptional regulation.

Structure/function analysis of the phosphatidylinositol-3-kinase domain of yeast tra1.

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A, and SRR3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of approximately 7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

The role of histone ubiquitylation and deubiquitylation in gene expression as determined by the analysis of an HTB1(K123R) Saccharomyces cerevisiae strain.

In Saccharomyces cerevisiae histone H2B is ubiquitylated at lysine 123 in a process requiring the E2-ubiquitin conjugase, Rad6. We have analyzed gene expression in a strain containing a variant of histone H2B with lysine 123 converted to arginine to address the mechanisms by which ubiquitylation and deubiquitylation of histone H2B affect gene expression. The SAGA complex component, Ubp8, is one of two proteases that remove the ubiquitin moiety at lysine 123. We show that changes in gene expression observed upon deletion of ubp8 are suppressed by htb1 ( K123R ), which provides genetic evidence that Ubp8 alters gene expression through deubiquitylation of histone H2B. Microarray analyses of the htb1 ( K123R ) strain show that loss of histone ubiquitylation results in a twofold or greater change in expression of approximately 1.5% of the protein coding genes with approximately 75% of these increasing. For genes in which ubiquitylation represses expression, ubiquitylation principally acts through its effects on histone methylation. In contrast, decreased expression of the CWP1 gene was not paralleled by deletions of methyltransferase components and is thus likely independent of methylation. Finally, by comparing gene expression changes in the htb1 ( K123R ) strain with those in a strain deleted for rad6, we conclude that lysine 123 affects transcription primarily because of it being a site of ubiquitylation.

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.