Fluorescently labeled recombinant RNAP system to probe archaeal transcription initiation.
The transcriptional apparatus is one of the most complex cellular machineries and in order to fully appreciate the behavior of these protein-nucleic acid assemblies one has to understand the molecular details of the system. In addition to classical biochemical and structural studies, fluorescence-based techniques turned out as an important--and sometimes the critical--tool to obtain information about the molecular mechanisms of transcription. Fluorescence is not only a multi-modal parameter that can report on molecular interactions, environment and oligomerization status. Measured on the single-molecule level it also informs about the heterogeneity of the system and gives access to distances and distance changes in the molecular relevant nanometer regime. A pre-requisite for fluorescence-based measurements is the site-specific incorporation of one or multiple fluorescent dyes. In this respect, the archaeal transcription system is ideally suited as it is available in a fully recombinant form and thus allows for site-specific modification via sophisticated labeling schemes. The application of fluorescence based approaches to the archaeal transcription apparatus changed our understanding of the molecular mechanisms and dynamics that drive archaeal transcription and unraveled the architecture of transcriptional complexes not amenable to structural interrogation.
Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Straße 10, 38106 Braunschweig, Germany.
Sarah
S
Schulz
Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Straße 10, 38106 Braunschweig, Germany.
Kevin
K
Kramm
RNAP Laboratory, University College London, Institute of Structural and Molecular Biology, Division of Biosciences, Gower St, London WC1E 6BT, UK.
Finn
F
Werner
Physikalische und Theoretische Chemie - NanoBioSciences, Technische Universität Braunschweig, Hans-Sommer-Straße 10, 38106 Braunschweig, Germany. Electronic address: d.grohmann@tu-braunschweig.de.
Dina
D
Grohmann
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012321:DNA-Directed RNA Polymerases
Archaea; Fluorescence; RNA polymerase; Single-molecule; Transcription