Transcriptional transactivation by selected short random peptides attached to lexA-GFP fusion proteins

BACKGROUND: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides. RESULTS: We performed a deliberate search of random peptide libraries for peptides capable of conferring transcriptional transactivation on the lexA DNA binding domain. Two libraries, one composed of C-terminal fusions, the other of peptide insertions within the green fluorescent protein structure, were used. We show that (i) peptide sequences other than C-terminal fusions can confer transactivation; (ii) though acidic activator peptides are more common, charge neutral and basic peptides can function as activators; and (iii) peptides as short as 11 amino acids behave in a modular fashion. CONCLUSIONS: These results support the recruitment model of transcriptional activation and, combined with other studies, suggest the possibility of using activator peptides in a variety of applications, including drug development work

In vitro analysis of the interaction between yeast fimbrin and actin: A model for a mechanism of allele-specific suppression

The actin cytoskeleton in yeast plays a role in many morphological events such as cell growth, secretion, polarity, bud emergence, and endocytosis. Some proteins have been identified that regulate the elongation, nucleation, and stability of the actin cytoskeleton. One of the many proteins controlling the state of the actin cytoskeleton is an actin bundling protein encoded by the gene SAC6 (suppressor of actin). In this study, I have examined some of the phenotypic consequences of overexpression of Sac6p and analyzed interactions between Sac6p and actin in vitro. I have investigated the molecular basis of allele-specific suppression observed between combination of act1 mutations and sac6 mutations by analyzing in vitro the interaction between wild-type and mutant actin and wild-type and mutant Sac6p. The biochemical assays show in vitro suppression of binding and crosslinking activity between Sac6 suppressor protein and mutant actin protein. In addition, the Sac6 suppressor proteins tested have an increased affinity to wild-type actin. This analysis, as well as previous genetic data, is consistent with the idea that suppression results from an overall increase in affinity to actin rather than a strict "lock and key" mechanism previously hypothesized. Overexpression of Sac6p under the control of a galactose inducible promoter results in growth inhibition, accumulation of multinucleated cells, and altered actin cytoskeletal organization. To better understand why overexpression of Sac6p has detrimental effects, I devised a screen to isolate genomic mutations that can suppress the growth defects resulting from elevated levels of Sac6p. I have found that an act1-3 strain is able to suppress Sac6p overexpression and one of the mutants isolated in the screen is also an act1 mutant allele. In addition, the temperature sensitive and osmotic sensitive phenotypes are not complemented by an act1-3 strain. These results suggest that the lethality is mediated through the interaction of Sac6p with actin. In addition, this analysis shows that the proper stoichiometry of Sac6p is critical for proper function of actin in vivo

Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.

Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other