Synergistic activation of transcription by the mutant and wild-type minimal transcriptional activation domain of VP16.
VP16 activates transcription by stimulating initiation, and for this function the aromatic residue at position 442 within its activation domain is critical. Recent studies have suggested that VP16 also stimulates transcriptional elongation. It has been shown that VP16 can activate transcription tethered downstream of the transcription start site to RNA. Here, we analyze the synergistic activation features of hybrid VP16 fusion proteins when tethered simultaneously to RNA downstream of the start site and to DNA upstream of a promoter in order to investigate its role in postinitiation control of transcription. Upon targeting the VP16 activation domain simultaneously to both DNA and RNA, high levels of transcriptional synergism is observed. Importantly, a transcription-defective VP16 minimal activation domain (amino acids 413-453) mutated at critical residue 442 (phenylalanine --> proline) maintained synergism, when bound to RNA, with the DNA-bound wild-type VP16 minimal activation domain. Targeting of this "functionally defective" VP16 minimal activation domain via RNA and an intact activation domain via DNA allowed us to uncover a postinitiation activity for VP16 not previously detected in DNA targeting studies. We suggest that, in addition to stimulating initiation, VP16 also acts at a postinitiation step involving residues other than the critical residue at position 442 within the same 41-amino acid minimal activation domain (amino acids 413-453) required for initiation.
Ghosh
S
S
Center for Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 78245-3207, USA.
Toth
C
C
Peterlin
B M
BM
Seto
E
E
D000595:Amino Acid Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry; D016325:HIV Long Terminal Repeat; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D012333:RNA, Messenger; D015534:Trans-Activators