Identification of eukaryotic promoter regulatory elements using nonhomologous random recombination.
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.
Doyon
Jeffrey B
JB
Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Liu
David R
DR
D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D016384:Consensus Sequence; D005656:Fungal Proteins / Q000235:genetics; D015723:Gene Library; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D015533:Transcriptional Activation
D050976:Basic-Leucine Zipper Transcription Factors; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D018840:HSP70 Heat-Shock Proteins; C060153:KAR2 protein, yeast; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; C513385:SIL1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins