Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex.
Induction of transcription of the immediate-early (IE) genes of herpes simplex virus involves the assembly of a DNA-binding complex containing the viral protein Vmw65 and the cellular transcription factor Oct-1. We show that Oct-1 is not sufficient for complex formation and that another cellular factor(s) which is absolutely required for complex formation can be separated from Oct-1 under native conditions. We have purified this factor by approximately 100-fold using DNA-cellulose, ion-exchange and size-exclusion chromatographies. The assay used throughout the purification procedure follows the ability of the cellular factor to form a complex when added to purified Oct-1, Vmw65 and an IE specific DNA probe. The complex forming factor (CFF) had a sedimentation coefficient of about 4.4 S (i.e. molecular mass of about 70K, under non-denaturing conditions) and the polypeptide profile of highly purified CFF demonstrated two major species with molecular masses of 80K and 70K. Unequivocal association of either of these two species with CFF activity could not presently be demonstrated due to the sensitivity of CFF to denaturation. CFF, when tested on its own or in the presence of Vmw65, did not bind to the IE-specific consensus motif. We have also used deletion mutants of Oct-1 to show that the POU domain of this protein was sufficient for CFF-dependent complex formation with Vmw65. Deletions of the POU specific region of Oct-1 significantly reduced the complex forming ability, although detectable levels of complex were reconstituted using Vmw65, CFF and just the homeodomain of Oct-1.
Katan
M
M
Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.
Haigh
A
A
Verrijzer
C P
CP
van der Vliet
P C
PC
O'Hare
P
P
D000818:Animals; D001483:Base Sequence; D002498:Centrifugation; D002852:Chromatography, Ion Exchange; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins