In vivo mutational analysis of the NGFI-A zinc fingers.
NGFI-A is a mammalian transcription factor that contains zinc fingers similar to those observed in several other proteins, including NGFI-C and Krox-20. To define precisely the DNA binding domain of NGFI-A, we selected mutants using a chimeric transcriptional activator that contains the NGFI-A zinc finger domain sandwiched between the lexA DNA binding domain and the GAL4 transcriptional activating domain. Expression of this lexA-NGFI-A-GAL4 (LAG) trimeric protein in yeast significantly retarded their growth, unlike an activator containing only the lexA and GAL4 components. This suggested that LAG inappropriately regulates genes in yeast that contain NGFI-A binding sites. Yeast that contained LAG reverted to wild-type growth at high frequency by inactivation of LAG. The mutations recovered from these revertants were specifically limited to the 83-residue NGFI-A zinc finger domain by requiring that the lexA and GAL4 portions of the LAG chimera remain functional. Nearly all of the 93 mutants obtained contained single missense mutations that mapped within the zinc fingers to residues thought to be important for zinc finger function. Deletion analysis of native NGFI-A verified that residues distant from the zinc fingers do not influence DNA binding, thus establishing the minimal functional DNA binding domain. Interestingly, many zinc finger residues ascribed specific functions by x-ray crystallography were never mutated in yeast, implying that the identity of these residues is not critical. Surprisingly, not all of the mutations tested significantly impaired NGFI-A-specific DNA binding, suggesting that the function of these zinc fingers is more diverse than previously recognized.
Wilson
T E
TE
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Day
M L
ML
Pexton
T
T
Padgett
K A
KA
Johnston
M
M
Milbrandt
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA