Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences

Ikeda, Richard A.; Chang, Lisa L.; Warshamana, G. Sakuntala

doi:10.1021/bi00086a016

Cited by 13 publications

(16 citation statements)

References 31 publications

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“…Previous studies have demonstrated the ability to screen for functional mutant polymerases in E. coli (29,30). In this way, a mutant T7 RNAP was identified that directs transcription from several different promoter sequences that are not utilized by the wild-type enzyme (29). It would be difficult to use the same strategy to select for T7 RNAP variants that utilize an all-RNA promoter.…”

Section: Discussionmentioning

confidence: 99%

Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

McGinness

Joyce

2002

Journal of Biological Chemistry

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A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the nontemplate and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and ␤-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that use T7 RNA polymerase for in vitro transcription. T7 RNA polymerase (T7 RNAP)1 is widely utilized in molecular biology research for the transcription of RNA. The behavior of this enzyme is critically dependent on its interaction with the promoter element. The T7 bacteriophage genome contains a variety of promoters that are recognized by T7 RNAP, all of which are related to a 23-base pair consensus sequence (see Fig. 1) (1). The promoter can be divided into a recognition domain, encompassing positions Ϫ17 through Ϫ5, and an initiation domain, encompassing positions Ϫ4 through ϩ6 (2-5). Transcription initiates at the ϩ1 position. Thus, the promoter is commonly viewed as spanning positions Ϫ17 through Ϫ1 even though the consensus sequence extends 6 base pairs beyond the site of initiation. In practice, this allows for the introduction of a wide variety of template sequences downstream of the Ϫ1 position to transcribe almost any desired RNA sequence.Numerous studies have illuminated details of the interactions between T7 RNAP and the recognition and initiation domains of the promoter. Through the use of Fe(II)-EDTA footprinting analysis it was shown that the protein recognizes the double-stranded DNA promoter on one face of the helix as indicated by protein protections that alternated between the template and nontemplate strands from positions Ϫ17 through ϩ2 (6). More recent studies involved the introduction of modified bases at defined locations within the promoter, providing information concerning interactions between T7 RNAP and specific functional groups of the DNA (4, 5, 7, 8). Those studies indicated major groove interactions in the recognition domain of the promoter at positions Ϫ11 through Ϫ5, confirming earlier modification interference experiments that implicated positions Ϫ12 through Ϫ5 as being important for binding interactions in the major groove of the DNA (9).The recent crystal structure of T7 RNAP complexed with its promoter confirmed the results of prior biochemical studies and supports a model in which polymerase binding occurs on one face of the DNA helix (10). A detailed picture of polymerasepromoter interactions is provided by this structure. Specifically, the AT-rich recognition loop of the polymerase (residues 93-101) lies...

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Section: Discussionmentioning

confidence: 99%

Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

McGinness

Joyce

2002

Journal of Biological Chemistry

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“…T7 RNAP is highly specific for its promoter sequence (TAATACGACTCACTATA), and exhibits virtually no detectable activity on the consensus promoter of the related bacteriophage T3 ( A A T TA ACC CTCACTA A A, differences underlined) 21,22. Despite decades of study and several attempts to engineer the specificity of T7 RNAP towards other promoters22,23 including that of T3, a mutant T7 RNAP capable of recognizing the T3 promoter has not been previously reported.…”

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confidence: 99%

A system for the continuous directed evolution of biomolecules

Esvelt

Carlson

Liu

2011

Nature

586

639

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Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention.1 Since evolutionary success is dependent on the total number of rounds performed,2 a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness.3 While researchers have accelerated individual steps in the evolutionary cycle,4–9 the only previous example of continuous directed evolution was the landmark study of Joyce,10 who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in E. coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerases that recognize a distinct promoter, initiate transcripts with A instead of G, and initiate transcripts with C. In one example, PACE executed 200 rounds of protein evolution over the course of eight days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than one week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

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“…Thus, the result confi rms a central rule of directed evolution approaches: 'What you get is what you screen for' [62]. The DNA-dependent RNA polymerase of bacteriophage T7 is an ideal target for directed evolution studies because it is a single-subunit enzyme that has been extensively characterized by mutational [63][64][65][66][67][68][69] and structural studies [70][71][72][73]. Like other phage RNA polymerases, the enzyme is highly specifi c for its promoter.…”

Section: Tuning Polymerase Activity By Directed Evolutionmentioning

confidence: 99%

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function

Brakmann

2005

Cell. Mol. Life Sci.

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Polynucleotide polymerases play a crucial role in transmitting genetic information from generation to generation, and they are the most important reagents in biotechnology. Although classical crystal structure analyses as well as biochemical studies have significantly contributed to our understanding of how DNA polymerases function, surprising new insights regarding the importance of certain residues and protein motifs, or of their mutability have been achieved in recent years by evolutionary approaches. Directed evolution has also facilitated the generation of polymerases with tailored substrate repertoires or with stabilities and activities beyond those of their naturally evolved counterparts. Recent new insights in polymerase structure-function relationships and new achievements in the development of tailored polymerases for current methods of nucleic acid synthesis will be summarized in this article.

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Selection and characterization of a mutant T7 RNA polymerase that recognizes an expanded range of T7 promoter-like sequences

Cited by 13 publications

References 31 publications

Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

A system for the continuous directed evolution of biomolecules

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function

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