Modulating the potency of an activator in a yeast in vitro transcription system.
The intrinsic stimulatory potential or potency of a eukaryotic gene activator is controlled by the interaction between the activation domain and the transcriptional machinery. To further understand this interaction, we undertook a biochemical study to identify parameters that could be used to modulate activator potency. We considered how varying the number of activation domains, their flexibility, and the number of promoter sites affects potency in a yeast nuclear extract. The effects of GAL4 derivatives bearing either one, two, or four herpes simplex virus VP16 activation domains (amino acids 413 to 454) were measured on DNA templates containing one or two GAL4 sites in a Saccharomyces cerevisiae nuclear extract. We found that multimerized VP16 activation domains acted synergistically to increase the potency of the activators. The spacing between the activation domains was critical, such that the increased flexibility imparted by a protein linker contributed to increased activator potency. With highly potent activators, the levels of transcription stimulated on a single site were saturating, whereas the stimulatory effect of weaker activators increased with the number of sites. We discuss how these biochemical studies relate to the mechanism of gene activation and synergy in a yeast in vitro system.
Ohashi
Y
Y
Department of Biological Chemistry, School of Medicine, University of California at Los Angeles 90024-1737.
Brickman
J M
JM
Furman
E
E
Middleton
B
B
Carey
M
M
D002467:Cell Nucleus / Q000378:metabolism; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017405:Herpes Simplex Virus Protein Vmw65 / Q000302:isolation & purification / Q000378:metabolism*; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018139:Simplexvirus / Q000378:metabolism; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors