The DNA binding domain of the vaccinia virus early transcription factor small subunit is an extended helicase-like motif.
The vaccinia virus early transcription factor (VETF) is an ATP-dependent activator of the early class of viral genes. VETF is a heterodimeric protein that binds an initiator-like element surrounding the start site of transcription. Previous studies indicated that the small subunit of VETF contacts the promoter DNA. We have taken a mutational approach to determine sequences in the VETF small subunit that are important for DNA binding. Two types of sequences were targeted for mutation: ones resembling motifs that are conserved in the nucleic acid helicase family and positively charged residues in predicted alpha-helices. Mutations affecting transcription activation were clustered in two regions. One mutation that impaired DNA binding is located near the N-terminus within the putative ATP-binding pocket that comprises helicase domain I. DNA binding was also severely reduced by mutations in a sequence resembling helicase domain VI and two putative alpha-helices that flank this domain in the C-terminal third of the polypeptide. These results indicate that the DNA binding domain in the small subunit of VETF is not isolated within a separable domain as is the case with most transcription factors, but rather, spans most of the length of the 637 residue polypeptide. A model for VETF structure is suggested in which the active site for ATP hydrolysis is integrated within an extended DNA-binding domain such that the structure and function of each domain influences that of the other.
Li
J
J
Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153, USA.
Broyles
S S
SS
D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA; D004265:DNA Helicases