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Unusual Sp1-GC box interaction in a parvovirus promoter
1Department of Molecular Biophysics, Yale University School of Medicine, New Haven, Connecticut 06510-8005.
Abstract:
The P4 promoter of the parvovirus minute virus of mice contains a single degenerate GC box sequence which binds the transcription factor Sp1 with high affinity. The two promoters of murine Sp1 were affinity purified, and their interactions with the P4 promoter were examined. Several unusual features were observed. Methylation interference experiments demonstrated that Sp1 makes contacts with both DNA strands, including the central guanine as well as an adenine residue on the cytidine-rich strand of the GC box. UV photocross-linking revealed that the 95- and the 105-kDa promoters cross-link exclusively to opposite strands of the GC box. These results suggest that the phosphorylation of the 95-kDa Sp1 promoter results in a change in the way Sp1 is positioned on the P4 GC box and identifies a high-affinity GC box motif.
Insights
Transcription factor Sp1 binds the minute virus of mice P4 promoter's GC box. Phosphorylation alters Sp1 positioning, revealing a high-affinity binding motif and DNA strand interactions.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The P4 promoter of the minute virus of mice (MVM) is crucial for viral gene expression.
- This promoter contains a degenerate GC box that binds the transcription factor Sp1 with high affinity.
Purpose of the Study:
- To investigate the interaction between the transcription factor Sp1 and the MVM P4 promoter.
- To elucidate the binding characteristics and DNA contacts of Sp1 on the P4 GC box.
Main Methods:
- Affinity purification of murine Sp1.
- Methylation interference assays to determine DNA-protein contacts.
- UV photocross-linking to identify DNA strand interactions.
Main Results:
- Sp1 binds to both DNA strands of the GC box, including key guanine and adenine residues.
- Different molecular weight forms of Sp1 (95-kDa and 105-kDa) cross-link to opposite DNA strands.
- Phosphorylation of the 95-kDa Sp1 influences its binding position on the P4 GC box.
Conclusions:
- Sp1 exhibits complex binding interactions with the MVM P4 promoter GC box.
- Phosphorylation plays a role in modulating Sp1's DNA binding orientation.
- These findings define a high-affinity GC box motif critical for parvovirus transcription.