title: Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid.
abstract: Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden.
authors: Arash
authors: A
authors: Sanamrad
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden.
authors: Fredrik
authors: http://orcid.org/0000-0002-5019-4312
authors: F
authors: Persson
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden.
authors: Ebba G
authors: EG
authors: Lundius
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden.
authors: David
authors: D
authors: Fange
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden.
authors: Arvid H
authors: AH
authors: Gynnå
authors: Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden johan.elf@icm.uu.se.
authors: Johan
authors: J
authors: Elf
mesh_terms: D058948:Cell Tracking / Q000379:methods*; D004269:DNA, Bacterial / Q000378:metabolism*; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D044085:Microfluidics; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D054657:Ribosome Subunits / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D004917:Erythromycin
keywords: antibiotics; nucleoid exclusion; single-molecule imaging; single-molecule tracking; transcription-translation coupling