Abstract
We used ribosome profiling to characterize the biological role of ribosome recycling factor (RRF) in Escherichia coli. As expected, RRF depletion leads to enrichment of post-termination 70S complexes in 3'-UTRs. We also observe that elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons. Previous studies have suggested a role for recycling in translational coupling within operons; if a ribosome remains bound to an mRNA after termination, it may re-initiate downstream. We found, however, that RRF depletion did not significantly affect coupling efficiency in reporter assays or in ribosome density genome-wide. These findings argue that re-initiation is not a major mechanism of translational coupling in E. coli. Finally, RRF depletion has dramatic effects on the activity of ribosome rescue factors tmRNA and ArfA. Our results provide a global view of the effects of the loss of ribosome recycling on protein synthesis in E. coli.
Highlights
After the synthesis of a protein is complete, the ribosomal subunits are separated from each other and from mRNA to be reused in the round of translation, a process known as ribosome recycling (Janosi et al, 1996)
Because ribosome recycling factor (RRF) is encoded by an essential gene, previous analyses of the in vivo function of this factor have relied on a temperature-sensitive mutant whose abundance drops significantly at elevated temperatures (43 ̊C) (Janosi et al, 1998)
We swapped the promoter in the genome with the araBAD promoter so that cells cultured in media with arabinose express RRF, but following the switch to media with glucose, they strongly repress its transcription
Summary
After the synthesis of a protein is complete, the ribosomal subunits are separated from each other and from mRNA to be reused in the round of translation, a process known as ribosome recycling (Janosi et al, 1996) This process always involves the active dissociation of post-termination complexes (post-TCs), the molecular mechanism of recycling differs among the three domains of life (Youngman et al, 2008; Buskirk and Green, 2017). The bacterial release factors RF1 and RF2 share similar names with their eukaryotic counterparts, they are evolutionarily unrelated and act alone to release the nascent peptide (Scolnick et al, 1968) Removal of these factors by the translational GTPase RF3 clears the way for binding of ribosome recycling factor (RRF) (Freistroffer et al, 1997; Peske et al, 2014; Koutmou et al, 2014). Binding of IF3 excludes the deacylated tRNA from the 30S subunit and prevents reassembly of the 70S complex (Prabhakar et al, 2017)
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