Abstract
Transcription factors (TFs) are cytoplasmic proteins that play an essential role in gene expression. These proteins form multimers and this phenomenon is thought to be one of the mechanisms that regulate transcription. TF molecules reach their DNA binding sites through the large central channel of the nuclear pore complex (NPC). However, the NPC channel is known to restrict the translocation of molecules ⩾20–70 kD. Therefore, during their translocation, TF molecules and/or their multimers may plug the NPC channel and thus, interrupt ion flow through the channel, with a concomitant reduction in the ion conductance of the channel (γ). Here we show with patch clamp that γ is reduced during translocation of three major TFs: c-Jun (40 kD), NF-κB (≈50 kD), and SP1 (≈100 kD). Within a minute, femtomolar concentrations of these proteins reduced γ suggesting a purely mechanical interaction between single TF molecules and the inner wall of the NPC channel. NPCs remained plugged for 0.5–3 hr in the absence of ATP but when ATP was added, channel plugging was shortened to <5 min. After unplugging, channel closures were rarely observed and the number of functional channels increased. The transcription factors also stabilized the NPCs as shown by the extended duration of the preparations which allowed recordings for up to 72 hr. These observations are the first direct demonstration of the important role of NPCs in mediating nuclear translocation of TFs and, therefore, in forming part of the mechanisms regulating gene expression. The studies also demonstrate the potential of the patch clamp technique in quantifying TF translocation to the nucleus, mRNA export, and other processes governing gene expression.
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Supported by grants from the American Heart Association, Maryland Affiliate, to JOB, from the Deutsche Forschungsgemeinschaft, SFB 176 (A 6) to HO, from National Institutes of Health Intramural Funding to JAH, and from the Medical Research Council of Canada to AL. Thanks are tendered to Peggy Kopps and the Technical Department of Promega for providing unpublished information on the transcriptions factors.
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Bustamante, J.O., Oberleithner, H., Hanover, J.A. et al. Patch clamp detection of transcription factor translocation along the nuclear pore complex channel. J. Membarin Biol. 146, 253–261 (1995). https://doi.org/10.1007/BF00233945
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DOI: https://doi.org/10.1007/BF00233945