Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo.
We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.
Gauthier-Rouvière
C
C
Department of Cell Biology, CRBM-CNRS/INSERM, Montpellier, France.
Caï
Q Q
QQ
Lautredou
N
N
Fernandez
A
A
Blanchard
J M
JM
Lamb
N J
NJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D006131:Growth Inhibitors; D008845:Microinjections; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000008:administration & dosage; D026362:Serum Response Factor; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology
D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D011994:Recombinant Proteins; D026362:Serum Response Factor; D014157:Transcription Factors