Abstract
The bacterial final sigma(54) protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent. Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes. Here we report that a fragment of Escherichia coli final sigma(54) encompassing amino acid residues 29-177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-final sigma(54) is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-final sigma(54) functions by a normal mechanism of transcriptional activation. Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-final sigma(54)-mediated activation and enhances the toxicity of Gal4-final sigma(54). Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-final sigma(54). Gal4-final sigma(54) activation and toxicity are also dependent upon specific final sigma(54) residues that are required for activator-responsive promoter melting by final sigma(54) in bacteria, implying that activation is a consequence of final sigma(54)-specific features rather than a structurally fortuitous polypeptide fragment. As such, Gal4-final sigma(54) represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.
Highlights
Promoter specificity of bacterial RNA polymerases (RNAPs)1 is conferred by sigma factors that bind the core RNAP to generate the ␣2Ј holoenzyme
Activation is dependent upon TFIIB residues that interact with RNA polymerase II (RNAP II)/TFIIF, suggesting that when tethered to promoter DNA, TFIIB stimulates transcription by recruiting the RNAP II holoenzyme complex; alternatively, TFIIB is a component of the holoenzyme complex such that the holoenzyme is recruited to the promoter by the
In this study we define a fragment of E. coli 54 that functions as a potent transcriptional activator when fused to a Gal4 DNA binding domain in yeast
Summary
Promoter specificity of bacterial RNA polymerases (RNAPs)1 is conferred by sigma factors that bind the core RNAP to generate the ␣2Ј holoenzyme. We report that a fragment of Escherichia coli 54 encompassing amino acid residues 29 –177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-54 is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-54 functions by a normal mechanism of transcriptional activation.
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