A combined in vitro / in vivo selection for polymerases with novel promoter specificities

被引：21

作者：

Chelliserrykattil J. ^{[1
]}

Cai G. ^{[1
]}

Ellington A.D. ^{[1
]}

机构：

[1] Department of Chemistry/Biochemistry, Inst. for Cellular/Molecular Biology, University of Texas at Austin, Austin, TX

来源：

BMC Biotechnology | / 1卷 / 1期

关键词：

Amino Acid Position; Promoter Specificity; Single Base Pair Substitution; Promoter Library; N748D Substitution;

D O I：

10.1186/1472-6750-1-13

中图分类号：

学科分类号：

摘要：

Background: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro / in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (103 - 106) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendent mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection. Results and Conclusions: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides. © 2001 Chelliserrykattil et al; licensee BioMed Central Ltd.

引用

页数：7

共 19 条

[1]

Cheetham G.M., Steitz T.A., Structure of a transcribing T7 RNA polymerase initiation complex, Science, 286, pp. 2305-2309, (1999)

[2]

Cheetham G.M., Jeruzalmi D., Steitz T.A., Structural basis for initiation of transcription from an RNA polymerase-promoter complex, Nature, 399, pp. 80-83, (1999)

[3]

Diaz G.A., Raskin C.A., McAllister W.T., Hierarchy of base-pair preference in the binding domain of the bacteriophage T7 promoter, J. Mol. Biol., 229, pp. 805-811, (1993)

[4]

Schick C., Martin C.T., Tests of a model of specific contacts in T7 RNA polymerase-promoter interactions, Biochemistry, 34, pp. 666-672, (1995)

[5]

Li T., Ho H.H., Maslak M., Schick C., Martin C.T., Major groove recognition elements in the middle of the T7 RNA polymerase promoter, Biochemistry, 35, pp. 3722-3727, (1996)

[6]

Raskin C.A., Diaz G., Joho K., McAllister W.T., Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity, J. Mol. Biol., 228, pp. 506-515, (1992)

[7]

Rong M., He B., McAllister W.T., Durbin R.K., Promoter specificity determinants of T7 RNA polymerase, Proc. Natl. Acad. Sci. USA, 95, pp. 515-519, (1998)

[8]

Ikeda R.A., Chang L.L., Warshamana G.S., Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences, Biochemistry, 32, pp. 9115-9124, (1993)

[9]

Raskin C.A., Diaz G.A., McAllister W.T., RNA polymerase mutants with altered promoter specificities, Proc. Natl. Acad. Sci. U. S. A., 90, (1993)

[10]

Dubendorff J.W., Studier F.W., Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter, J. Mol. Biol., 219, pp. 61-68, (1991)

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