Promoter analysis by saturation mutagenesis
Biological Procedures Online volume 3, pages 64–69 (2001)
Abstract
Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.
Abbreviations
- bop :
-
bacterioopsin
References
Baliga NS, DasSarma S. Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter. J Bacteriol. 1999;181(8):2513–2518.
Baliga NS, Dassarma S. Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity. Mol Microbiol. 2000;36(5):1175–1183.
Barik S, Galinski MS. “Megaprimer” method of PCR: increased template concentration improves yield. Biotechniques. 1991;10(4):489–490.
Blattner FR. Direct amplification of the entire ITS region from poorly preserved plant material using recombinant PCR. Biotechniques. 1999;27(6):1180–1186.
Chrzanowska-Lightowlers ZM, Temperley RJ, McGregor A, Bindoff LA, Lightowlers RN. Conversion of a reporter gene for mitochondrial gene expression using iterative mega-prime PCR. Gene. 1999;230(2):241–247.
Colgan J, Manley JL. Cooperation between core promoter elements influences transcriptional activity in vivo. Proc Natl Acad Sci U S A. 1995;92(6):1955–1959.
Corona V, Aracri B, Kosturkova G, et al. Regulation of a carotenoid biosynthesis gene promoter during plant development. Plant J. 1996;9(4):505–512.
Danner S, Soppa J. Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection. Mol Microbiol. 1996;19(6): 1265–1276.
Hain J, Reiter WD, Hudepohl U, Zillig W. Elements of an archaeal promoter defined by mutational analysis. Nucleic Acids Res. 1992;20(20):5423–5428.
Patenge N, Haase A, Bolhuis H, Oesterhelt D. The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum. Mol Microbiol. 2000;36(1):105–113.
Palmer JR, Daniels CJ. In vivo definition of an archaeal promoter. J Bacteriol. 1995;177 (7):1844–1849.
Reiter WD, Hudepohl U, Zillig W. Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro. Proc Natl Acad Sci U S A. 1990;87(24):9509–9513.
Yang CF, Kim JM, Molinari E, DasSarma S. Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure. J Bacteriol. 1996;178 (3):840–845.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published: December 22, 2001
Rights and permissions
About this article
Cite this article
Baliga, N.S. Promoter analysis by saturation mutagenesis. Biol Proced Online 3, 64–69 (2001). https://doi.org/10.1251/bpo24
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1251/bpo24