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.

[1]  R. Kornberg,et al.  Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro. , 1989, Science.

[2]  Kevin Struhl,et al.  Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein , 1988, Nature.

[3]  W. Schaffner,et al.  Different activation domains stimulate transcription from remote (‘enhancer’) and proximal (‘promoter’) positions. , 1992, The EMBO journal.

[4]  G. Schaffner,et al.  Redundancy of information in enhancers as a principle of mammalian transcription control. , 1988, Journal of molecular biology.

[5]  W. Schaffner,et al.  Transcription factor Oct‐2A contains functionally redundant activating domains and works selectively from a promoter but not from a remote enhancer position in non‐lymphoid (HeLa) cells. , 1990, The EMBO journal.

[6]  Michael R. Green,et al.  Mechanism of action of an acidic transcriptional activator in vitro , 1991, Cell.

[7]  J. Gralla,et al.  Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. , 1992, Science.

[8]  K. Struhl,et al.  Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Chasman,et al.  Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro , 1989, Molecular and cellular biology.

[10]  M Ptashne,et al.  Cooperative DNA binding of the yeast transcriptional activator GAL4. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  W. Herr,et al.  Differential transcriptional activation by Oct-1 and Oct-2: Interdependent activation domains induce Oct-2 phosphorylation , 1990, Cell.

[12]  J. Brickman,et al.  New eukaryotic transcriptional repressers , 1993, Nature.

[13]  Roger D. Kornberg,et al.  A novel mediator between activator proteins and the RNA polymerase II transcription apparatus , 1990, Cell.

[14]  K. Struhl,et al.  Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription , 1990, Molecular and cellular biology.

[15]  R. Schüle,et al.  Many transcription factors interact synergistically with steroid receptors. , 1988, Science.

[16]  M. Carey,et al.  A synergistic increase in potency of a multimerized VP16 transcriptional activation domain. , 1992, The EMBO journal.

[17]  S. McKnight,et al.  Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. , 1988, Genes & development.

[18]  J. Workman,et al.  Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: differential function of DNA-binding domains. , 1991, Genes & development.

[19]  Michael Carey,et al.  A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives , 1990, Nature.

[20]  R. Tjian,et al.  Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. , 1989, Science.

[21]  N. Webster,et al.  The human estrogen receptor has two independent nonacidic transcriptional activation functions , 1989, Cell.

[22]  M. Carey,et al.  Transcriptional synergy by the Epstein-Barr virus transactivator ZEBRA , 1992, Journal of virology.

[23]  R. Evans,et al.  Multiple and cooperative trans-activation domains of the human glucocorticoid receptor , 1988, Cell.

[24]  Mark Ptashne,et al.  Negative effect of the transcriptional activator GAL4 , 1988, Nature.

[25]  D. Herschlag,et al.  Synergism in transcriptional activation: a kinetic view. , 1993, Genes & development.

[26]  M. Ptashne,et al.  Activators and targets , 1990, Nature.

[27]  I. Herskowitz,et al.  Characterization of the yeast SWI1, SWI2, and SWI3 genes, which encode a global activator of transcription , 1992, Cell.

[28]  R. Kornberg,et al.  Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Jun Ma,et al.  Deletion analysis of GAL4 defines two transcriptional activating segments , 1987, Cell.

[30]  S. Triezenberg,et al.  Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  B. O’Malley,et al.  Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements , 1989, Cell.

[32]  W. D. Cress,et al.  Critical structural elements of the VP16 transcriptional activation domain. , 1991, Science.

[33]  Andrew J. Bannister,et al.  Conserved motifs in Fos and Jun define a new class of activation domain. , 1992, Genes & development.

[34]  Michael Carey,et al.  How different eukaryotic transcriptional activators can cooperate promiscuously , 1990, Nature.

[35]  R. Tjian,et al.  Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. , 1991, Genes & development.

[36]  Michael Carey,et al.  DNA recognition by GAL4: structure of a protein-DNA complex , 1992, Nature.

[37]  J. Workman,et al.  Nucleosome core displacement in vitro via a metastable transcription factor-nucleosome complex. , 1992, Science.