Nucleic Acids Research Advance Access originally published online on August 24, 2007
Nucleic Acids Research 2007 35(17):5851-5860; doi:10.1093/nar/gkm634
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Nucleic Acids Research, 2007, Vol. 35, No. 17 5851-5860
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Identification of eukaryotic promoter regulatory elements using nonhomologous random recombination
Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA
*To whom correspondence should be addressed. Tel: + 1 617 496 1067; Fax: + 1 617 496 5688; Email: drliu{at}fas.harvard.edu
Received May 17, 2007. Revised July 31, 2007. Accepted August 2, 2007.
Understanding the regulatory logic of a eukaryotic promoter requires the elucidation of the regulatory elements within that promoter. Current experimental or computational methods to discover regulatory motifs within a promoter can be labor intensive and may miss redundant, unprecedented or weakly activating elements. We have developed an unbiased combinatorial approach to rapidly identify new upstream activating sequences (UASs) in a promoter. This approach couples nonhomologous random recombination with an in vivo screen to efficiently identify UASs and does not rely on preconceived hypotheses about promoter regulation or on similarity to known activating sequences. We validated this method using the unfolded protein response (UPR) in yeast and were able to identify both known and potentially novel UASs involved in the UPR. One of the new UASs discovered using this approach implicates Crz1 as a possible activator of Hac1, a transcription factor involved in the UPR. This method has several advantages over existing methods for UAS discovery including its speed, potential generality, sensitivity and lack of false positives and negatives.