Crystallization and characterization of PU.1/IRF-4/DNA ternary complex.
PU.1 and IRF-4, members of the Ets and interferon regulatory families of transcription factors, respectively, form a cooperative ternary complex that is implicated in the regulation of B-cell-specific gene expression. A portion of the cooperativity involves interaction between the PU.1 and IRF-4 DNA-binding domains. We report here the crystallization and preliminary characterization of PU.1 and IRF-4 DNA-binding domains bound to a 21-mer DNA site from the lambdaB element of immunoglobulin light chain lambda(2-4) enhancer. The crystals belong to space group P2(1) with unit cell dimensions of a=40.7A, b=62.3A, c=67.9A, beta=95.6 degrees with one complex molecule per asymmetric unit. Crystals diffract to a resolution of 3A using X-rays from a rotating anode generator but improve to 2.3A with synchrotron radiation. The crystals are highly mosaic, but the mosaicity can be improved following a series of steps involving the addition of additives, use of peritone oil as a cryoprotectant, slow flash-cooling in the cryostream, and several cycles of crystal annealing.
Escalante
Carlos R
CR
Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA.
Shen
Leyi
L
Escalante
Mary C
MC
Brass
Abraham L
AL
Edwards
Thomas A
TA
Singh
Harinder
H
Aggarwal
Aneel K
AK
D000818:Animals; D001483:Base Sequence; D003451:Cryoprotective Agents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000378:metabolism; D050835:Interferon Regulatory Factors; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D017356:Synchrotrons; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry*; D014961:X-Ray Diffraction; D000099232:Proto-Oncogene Protein Spi-1; D000099218:Interferon Regulatory Factor-4
D003451:Cryoprotective Agents; D004247:DNA; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D050835:Interferon Regulatory Factors; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; D000099218:Interferon Regulatory Factor-4