A Novel Complex: A Quantum Dot Conjugated to an Active T7RNA Polymerase

Mette Eriksen,Szabolcs Semsey,Peter Horvath,Michael A Sørensen,Liselotte Jauffred,Lene B Oddershede

doi:10.1155/2013/468105

Mette Eriksen, Szabolcs Semsey + Show 4 more

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https://doi.org/10.1155/2013/468105

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Abstract

To perform single-molecule studies of the T7RNA polymerase, it is crucial to visualize an individual T7RNA polymerase, for example, through a fluorescent signal. We present a novel complex combining two different molecular functions, an active T7RNA polymerase and a highly luminescent nanoparticle, a quantum dot. The complex has the advantage of both constituents: the complex can traffic along DNA and simultaneously be visualized, both at the ensemble and at the single-molecule level. The labeling was mediated through anin vivobiotinylation of a His-tagged T7RNA polymerase and subsequent binding of a streptavidin-coated quantum dot. Our technique allows for easy purification of the quantum dot labeled T7RNA polymerases from the reactants. Also, the conjugation does not alter the functionality of the polymerase; it retains the ability to bind and transcribe.

Highlights

RNA polymerase (RNAP) is an enzyme that catalyses transcription
The T7 RNAPs were biotinylated in vivo with a procedure that parallels the method used for biotinylating a lambda phage subsequently labeled by a quantum dots (QDs) [29]
In order to purify the QD : T7RNAP complexes they were mixed with an avidin resin, with the goal of capturing possible BioT7 RNAPs which did not bind to QDs

Summary

Introduction

RNA polymerase (RNAP) is an enzyme that catalyses transcription. It shall balance two tasks: binding to specific sites on DNA where transcription is initiated (promoters) and the ability to disengage at the termination signals. The T7 family of RNAPs is structurally and evolutionary distinct from the multisubunit family of RNAPs. T7 RNAP allows specific high-level transcription and expression of cloned genes [4]; it is a key enzyme in the production of recombinant proteins and in several in vitro molecular biology applications [5, 6]. T7 RNAP allows specific high-level transcription and expression of cloned genes [4]; it is a key enzyme in the production of recombinant proteins and in several in vitro molecular biology applications [5, 6] Such applications include the generation of large quantities of RNA transcripts for translation, synthesis of tRNA, rRNA, RNA virus genomes, ribozymes, microarray targets [7], production of substrates for RNA splicing [8], RNA secondary structure, antisense RNA, and RNA-protein interaction studies. Tight regulation of T7 RNAP expression is possible and T7 systems are used for the production of otherwise deleterious protein products [10]

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