title: Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.
abstract: The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.
authors.affiliation: Cold Spring Harbor Laboratory, New York 11724.
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: W
authors.initials: W
authors.lastname: Herr
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050813:Octamer Transcription Factor-2; D015345:Oligonucleotide Probes / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D050813:Octamer Transcription Factor-2; D015345:Oligonucleotide Probes; C058435:POU2F2 protein, human; D014157:Transcription Factors