title: Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro.
abstract: The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances. In the same time RNAP can be put in various types of states, such as paused, roadblocked, backtracked, etc. The experimental system thus allows studying the effects of the ribosome on different aspects of transcription elongation and the effects by RNAP on translation.
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne NE2 4AX, UK. Electronic address: d.a.castro-roa@ncl.ac.uk.
authors.forename: Daniel
authors.initials: D
authors.lastname: Castro-Roa
authors.affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne NE2 4AX, UK.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Zenkin
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D008967:Molecular Biology / Q000379:methods; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases
keywords: RNA; Ribosome; Transcription; Transcription–translation coupling; Translation RNA polymerase