Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter
Name:
1-s2.0-S0021925820001714-main.pdf
Size:
2.562Mb
Format:
PDF
Description:
Final Published Version
Affiliation
Department of Chemistry and Biochemistry, University of ArizonaIssue Date
2020-12-10
Metadata
Show full item recordCitation
Kartje, Z. J., Janis, H. I., Mukhopadhyay, S., & Gagnon, K. T. (2021). Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter. Journal of Biological Chemistry, 296, 100175.Journal
Journal of Biological ChemistryRights
© 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Collection Information
This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.Abstract
Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro. © 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.Note
Open access articleISSN
0021-9258EISSN
1083-351XPubMed ID
33303627Version
Final published versionae974a485f413a2113503eed53cd6c53
10.1074/jbc.RA120.014553
Scopus Count
Collections
Except where otherwise noted, this item's license is described as © 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).