Ribosome recycling is not critical for translational coupling in Escherichia coli
- Department of Molecular Biology and Genetics, United States
- Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Strategy for ribosome recycling factor (RRF) depletion.
(A) In the genome, the promoter upstream of frr (the gene encoding RRF) was replaced by the ligand-inducible araBAD promoter. A FLAG tag and residues YALAA were added to facilitate RRF detection and …
Figure 1—figure supplement 1
Protein synthesis continues after ribosome recycling factor (RRF) knock down.
(A) Cells were grown in media containing HPG (a methionine analog containing an alkyne moiety) for a period of 10 min at various times after the second change to media containing glucose. (B) …
Figure 2
RRF knockdown creates queues of stalled ribosomes upstream of stop codons and high ribosome density in the 3′-UTR.
(A) Ribosome density (in reads per million mapped reads) on the rpsB gene 60 min after the 2nd media change in the wild-type (top) and RRF knock-down strain (bottom). (B–D) Average ribosome density …
Figure 3
Ribosome recycling factor (RRF) depletion does not upregulate translation of GFP in the 3′-UTR of two genes.
(A) The reporter plasmid encodes an E. coli gene with its native promoter, 5′-UTR and 3′-UTR. The GFP ORF is inserted into the 3′-UTR in each reading frame relative to the upstream ORF. The GFP-only …
Figure 4 with 1 supplement
Highly translated genes have more stacked ribosomes and less relative ribosome density in the 3′-UTR.
(A) High and low ribosome occupancy genes were selected using the ratio of ribosome profiling to RNAseq in the WT sample. Here, we show the average ribosome density aligned at stop codons using the …
Figure 4—figure supplement 1
Average ribosome density aligned at stop codons of genes with low and high ribosome occupancy.
(A) From high-salt wash ribosome profiling after 15 min. (B) From high-salt wash ribosome profiling after 60 min. (C) From standard ribosome profiling after 5 min. (D) From standard ribosome …
Figure 5
RRF depletion has little or no effect on five gene pairs known to be translationally coupled.
(A) Reporter plasmids encode both mCherry and nano-luciferase separated by the last 120 nt of the upstream gene, the intergenic region, and the first 120 nucleotides of the downstream gene. The RBS …
Figure 6
RRF depletion has little or no effect on the relative expression of gene pairs on polycistronic messages genome-wide.
(A) Gene models showing the ribosome density (in reads per million mapped reads) on the clpPX operon 60 min after the second media change in the wild type (top) and RRF knock-down strain (bottom). …
Figure 7
Ribosome recyclinf factor (RRF) depletion upregulates ArfA expression.
(A) Scatter plot of ribosome density (in rpkm) in the wild type (x-axis) and RRF knock-down strain (y-axis) 60 min after the second media change. Genes with 10-fold increase of ribosome density are …
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