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

Chelliserrykattil J, Cai G, Ellington AD - BMC Biotechnol. (2001)

Bottom Line: Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, USA. jijumon@mail.utexas.edu

ABSTRACT

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 (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant 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.

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Analysis of polymerase expression by SDS-PAGE. Cells containing autogene constructs were grown to an O.D600 of 0.4 and induced with IPTG. Aliquots of cells were lysed at time points following induction, and separated on SDS-PAGE. Bands were visualized using Coomassie blue. Arrows indicate the relative position of T7 RNA polymerase. The normalization of samples is apparent from the amount of E. coli proteins present in each lane. (a) Comparison of the activities of the Q758C polymerase on a mutant promoter (GACG at positions -11 to -8) and the wild-type promoter (GACT at positions -11 through -8). (b) Comparison of the activities of the R3-17 polymerase with three different promoters (GGTA, TATA, and TGTA at positions -11 through -8) and the activity of the wild-type autogene with its promoter (GACT at positions -11 through -8).
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Figure 2: Analysis of polymerase expression by SDS-PAGE. Cells containing autogene constructs were grown to an O.D600 of 0.4 and induced with IPTG. Aliquots of cells were lysed at time points following induction, and separated on SDS-PAGE. Bands were visualized using Coomassie blue. Arrows indicate the relative position of T7 RNA polymerase. The normalization of samples is apparent from the amount of E. coli proteins present in each lane. (a) Comparison of the activities of the Q758C polymerase on a mutant promoter (GACG at positions -11 to -8) and the wild-type promoter (GACT at positions -11 through -8). (b) Comparison of the activities of the R3-17 polymerase with three different promoters (GGTA, TATA, and TGTA at positions -11 through -8) and the activity of the wild-type autogene with its promoter (GACT at positions -11 through -8).

Mentions: Our combined in vitro / in vivo selection scheme was designed to foster the self-amplification of novel polymerase variants (Figure 1). The T7 RNA polymerase gene was linked to a T7 RNA polymerase promoter, creating a so-called autogene [10], whose activity can be initiated by the basal level expression of polymerase in the cell. Upon transformation into E. coli, the autogene engendered the production of large amounts of T7 RNA polymerase (Figure 2). However, when the polymerase was cloned adjacent to mutant T7 RNA polymerase promoters, little T7 RNA polymerase expression was observed. We reasoned that any polymerase variant that could recognize the mutant promoter would re-establish the feedback loop and concomitantly lead not only to high protein expression levels, but also to high mRNA expression levels. In consequence, mRNA extracted from a population of cells transformed with a polymerase library should represent polymerase variants in rough proportion to their ability to utilize the mutant promoter. These mRNAs could be amplified en masse, re-cloned, and re-transformed into E. coli. Multiple cycles of selection and amplification should ultimately lead to the accumulation of those polymerase variants that were most successful at facilitating their own expression.


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

Chelliserrykattil J, Cai G, Ellington AD - BMC Biotechnol. (2001)

Analysis of polymerase expression by SDS-PAGE. Cells containing autogene constructs were grown to an O.D600 of 0.4 and induced with IPTG. Aliquots of cells were lysed at time points following induction, and separated on SDS-PAGE. Bands were visualized using Coomassie blue. Arrows indicate the relative position of T7 RNA polymerase. The normalization of samples is apparent from the amount of E. coli proteins present in each lane. (a) Comparison of the activities of the Q758C polymerase on a mutant promoter (GACG at positions -11 to -8) and the wild-type promoter (GACT at positions -11 through -8). (b) Comparison of the activities of the R3-17 polymerase with three different promoters (GGTA, TATA, and TGTA at positions -11 through -8) and the activity of the wild-type autogene with its promoter (GACT at positions -11 through -8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC64648&req=5

Figure 2: Analysis of polymerase expression by SDS-PAGE. Cells containing autogene constructs were grown to an O.D600 of 0.4 and induced with IPTG. Aliquots of cells were lysed at time points following induction, and separated on SDS-PAGE. Bands were visualized using Coomassie blue. Arrows indicate the relative position of T7 RNA polymerase. The normalization of samples is apparent from the amount of E. coli proteins present in each lane. (a) Comparison of the activities of the Q758C polymerase on a mutant promoter (GACG at positions -11 to -8) and the wild-type promoter (GACT at positions -11 through -8). (b) Comparison of the activities of the R3-17 polymerase with three different promoters (GGTA, TATA, and TGTA at positions -11 through -8) and the activity of the wild-type autogene with its promoter (GACT at positions -11 through -8).
Mentions: Our combined in vitro / in vivo selection scheme was designed to foster the self-amplification of novel polymerase variants (Figure 1). The T7 RNA polymerase gene was linked to a T7 RNA polymerase promoter, creating a so-called autogene [10], whose activity can be initiated by the basal level expression of polymerase in the cell. Upon transformation into E. coli, the autogene engendered the production of large amounts of T7 RNA polymerase (Figure 2). However, when the polymerase was cloned adjacent to mutant T7 RNA polymerase promoters, little T7 RNA polymerase expression was observed. We reasoned that any polymerase variant that could recognize the mutant promoter would re-establish the feedback loop and concomitantly lead not only to high protein expression levels, but also to high mRNA expression levels. In consequence, mRNA extracted from a population of cells transformed with a polymerase library should represent polymerase variants in rough proportion to their ability to utilize the mutant promoter. These mRNAs could be amplified en masse, re-cloned, and re-transformed into E. coli. Multiple cycles of selection and amplification should ultimately lead to the accumulation of those polymerase variants that were most successful at facilitating their own expression.

Bottom Line: Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, USA. jijumon@mail.utexas.edu

ABSTRACT

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 (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant 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.

Show MeSH
Related in: MedlinePlus