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ISBN 9783843922586

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978-3-8439-2258-6, Reihe Biophysik

Julia Nagy
The complete architecture of the archaeal RNA polymerase open complex obtained from single-molecule FRET and NPS

161 Seiten, Dissertation Universität Ulm (2015), Softcover, A5

Zusammenfassung / Abstract

All cellular life forms are presently classified into a three-domain system consisting of Archaea, Bacteria and Eukarya. For decades, Archaea were misclassified as Bacteria due to their prokaryotic morphology and metabolic diversity. However, the archaeal information-processing systems are distinctly eukaryotic.

Transcription is the first step in gene expression, a process in which messenger RNA is synthesised from genetic information, carried out by multisubunit RNA polymerases (RNAPs). In contrast to Eukarya, Archaea utilise only one RNAP to transcribe their genes, but its structure and utilisation of general transcription factors is strikingly similar to the eukaryotic RNA polymerase II (Pol II) system. However, the only basal transcription factors that are essential for archaeal transcription initiation are the TATA box-binding protein (TBP) and transcription factor B (TFB). A third factor, transcription factor E (TFE) interacts with RNAP but is not strictly required.

The archaeal RNAP system offers substantial experimental advantages as it is possible to reconstitute the entire transcription system of the hyperthermophilic Archaea M. jannaschii from recombinant proteins in vitro under defined conditions. This enables the site-specific introduction of fluorescent dyes for single-molecule fluorescence analysis into separate RNAP subunits and transcription factors.

In this thesis, single-molecule Förster resonance energy transfer (smFRET) and the Bayesian parameter estimation based Nano-Positioning System (NPS) analysis were combined to determine the positions of the transcription factors and the non-template DNA (ntDNA) strand relative to the RNAP. This allowed to built a model of the architecture of the complete open complex (OC) composed of the promoter DNA, TBP, TFB, TFE and the RNAP, elucidating the path of the ntDNA and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the eukaryotic helicase encoding factor TFIIH.

The results allow a brief insight into the molecular mechanisms of transcription initiation, promoter escape and transcription elongation in Archaea.