title: Dissociation time from DNA determines transcriptional function in a STAT1 linker mutant.
abstract: The STAT1 transcription factor is organized into several highly conserved domains, each of which has been assigned a function with the exception of the linker domain. We previously characterized a mutant in the linker domain of STAT1 that gave normal DNA binding using a standard probe in an electrophoretic mobility assay but failed to activate transcription in response to interferon gamma. We now report the mechanistic basis for the inactivity of this STAT1(K544A/E545A) mutant. Rather than failing to attract transcriptional coactivators, the STAT1(K544A/E545A) mutant has a subtle biophysical defect, which prevents accumulation of the activated protein on chromatin in vivo: the mutant has comparable K(d) with greatly increased k(off) for DNA binding. The increase in both on-rate and off-rate of DNA binding results in a substantially reduced residence time of STAT1(K544A/E545A) on STAT binding sites. We find a similar correlation between off-rate and transcriptional potency for STAT1(N460A), which bears a mutation in the DNA binding domain. These results yield insight into the rate of complex assembly involving STAT1 that leads to transcriptional stimulation.
authors.affiliation: Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021-6399, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Yang
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Henriksen
authors.forename: Olaf
authors.initials: O
authors.lastname: Schaefer
authors.forename: Natalia
authors.initials: N
authors.lastname: Zakharova
authors.forename: James E
authors.initials: JE
authors.lastname: Darnell
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D018909:src Homology Domains
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D004247:DNA; D008156:Luciferases