Alteration of specific amino acid residues in the acidic domain I of VSV phosphoprotein (P) converts a GAL4-P(I) hybrid into a transcriptional activator.
As part of a study of transcriptional regulation by viral proteins, we examined whether an acidic region from a regulatory protein of an RNA virus could function as a trans-activator. The NH2-terminal highly acidic domain I of the phosphoprotein (P) of vesicular stomatitis virus (VSV) was fused to the DNA-binding domain of the yeast trans-activator, GAL4. In transient transfection assays, the resulting chimeric protein failed to activate transcription of a reporter CAT gene. However, mutation of basic amino acid residues located at positions 6 and 8 or the alteration of eight amino acids within the acidic domain to eight different amino acids converted the chimeric protein into a transcriptional activator comparable to wild-type GAL4. When subjected to SDS-polyacrylamide gel electrophoresis, the P proteins containing trans-activation-positive mutations in domain I showed an altered mobility, suggesting that these mutations may have caused a conformational change that is critical for trans-activation. Since the acidity of P domain I is not sufficient to activate transcription, additional features of this region must play an important role in GAL4-mediated trans-activation. None of the trans-activation-positive mutants supported VSV RNA transcription in vitro. These results suggest that the amino acid residues within P domain I that can be made to function in the trans-activation of DNA-dependent RNA transcription are distinct from those involved in VSV RNA-dependent RNA transcription.
Takacs
A M
AM
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195.
Perrine
K G
KG
Barik
S
S
Banerjee
A K
AK
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins