Old dogmas revisited: exploring the coupling of transcription and translation in bacteria

The coupling of transcription and translation (CTT) is widely accepted as a distinctive feature of bacterial gene expression. This tenet was established 50 years ago, when electron micrographs by Miller and coworkers showed ribosomes engaged in translation of nascent mRNAs, therefore forming an “RNAP-mRNA-ribosome” complex. However, the universality of CTT has been recently challenged by two lines of evidence: 1) the transcriptional and translational machinery are highly segregated in Escherichia coli and Bacillus subtilis; 2) mRNAs have been shown to escape tight translational coupling and to self-localize to discreet subcellular localizations that correlate with the localizations of their corresponding encoded proteins. In order to reconcile the CTT dogma with these apparently conflicting observations, we aim at measuring the extent of CTT in E. coli by CTT-RNAseq and CTT-ribosome profiling. Our results so far demonstrate that a protocol relying on fast-chilling of cells and formaldehyde cross-linking is able to simultaneously halt transcription and translation and stabilize the “RNAP-mRNA-ribosome” complexes. These tertiary complexes can be subsequently isolated by pulling down RNAP together with ribosomes engaged in CTT. Total RNA and ribosome footprints extracted from these samples are used to construct RNAseq libraries that reflect the actual in vivo extent of CTT at operon and codon resolution, respectively. Beyond merely measuring CTT occurrence, we expect our results to unmask mechanistic features mediating CTT and to address key questions regarding the involvement of CTT in transertion (coupled transcription-translation-membrane insertion), mRNA targeting and overall subcellular organization.