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Rational design of a super core promoter that enhances gene expression

Nature Methods volume 3, pages 917922 (2006) | Download Citation

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Abstract

Transcription is a critical component in the expression of genes. Here we describe the design and analysis of a potent core promoter, termed super core promoter 1 (SCP1), which directs high amounts of transcription by RNA polymerase II in metazoans. SCP1 contains four core promoter motifs—the TATA box, initiator (Inr), motif ten element (MTE) and downstream promoter element (DPE)—in a single promoter, and is distinctly stronger than the cytomegalovirus (CMV) IE1 and adenovirus major late (AdML) core promoters both in vitro and in vivo. Each of the four core promoter motifs is needed for full SCP1 activity. SCP1 is bound efficiently by TFIID and exhibits a high propensity to form productive transcription complexes. SCP1 and related super core promoters (SCPs) with multiple core promoter motifs will be useful for the biophysical analysis of TFIID binding to DNA, the biochemical investigation of the transcription process and the enhancement of gene expression in cells.

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Acknowledgements

We are grateful to C.Y. Lim, J.-Y. Hsu and D. Urwin for advice throughout this study. We thank J.-Y. Hsu, T. Yusufzai, B. Rattner, D. Urwin, D. Fyodorov, J. Theisen, T. Bretz and C.Y. Lim for critical reading of the manuscript. We thank C. Inouye and R. Tjian for providing the purified human TFIID, C.Y. Lim for the purified D. melanogaster TFIID, J.-Y. Hsu for the pGL3-Basic vector with a modified polylinker, E.-T. Wong and G. Wahl for the pOG33-SV40 β-gal cotransfection plasmid, and W. Cavenee and F. Furnari for the use of their luminometer. This work was supported by a grant from the US National Institutes of Health (GM041249) to J.T.K.

Author information

Affiliations

  1. Section of Molecular Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

    • Tamar Juven-Gershon
    • , Susan Cheng
    •  & James T Kadonaga

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Contributions

J.T.K. and T.J.-G. conceived the experimental design. T.J.-G. performed the experiments. S.C. prepared the HeLa nuclear extracts and generated the SCP1, CMV and AdML −36 to +45 core promoter pUC119 constructs used in the experiments in Figure 1. T.J.-G. and J.T.K. wrote the manuscript.

Competing interests

A patent application covering the work described in this article has been filed by The Regents of the University of California. Although the authors do not have a personal conflict of interest, the authors' employer, The Regents of the University of California, could potentially gain financially from the publication of the studies described in this work.

Corresponding author

Correspondence to James T Kadonaga.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    SCP1 is transcribed more efficiently than the CMV or AdML core promoters.

  2. 2.

    Supplementary Fig. 2

    Analysis of the binding of purified human TFIID to SCP1 as well as to SCP1 variants that contain mutations in the TATA box, Inr, MTE, or DPE.

  3. 3.

    Supplementary Fig. 3

    SCP1 is more active than the CMV or AdML core promoters in Chinese hamster ovary (CHO) cells.

  4. 4.

    Supplementary Fig. 4

    SCP1 is more active than SCP2 in the absence of an enhancer as well as with the SV40 enhancer.

  5. 5.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth937

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