Rho-dependent transcription termination within the topAI gene.

(A) Schematic representation of topAI-lacZ transcriptional reporter fusions, indicating the mutation in the putative rut. (B) β-galactosidase activity of topAI-lacZ transcriptional fusions in wild-type (strain AMD054) or rho mutant (R66S amino acid substitution in Rho; strain GB004) cells. Reporter fusions with wild-type sequence or mutation of the predicted rut (rut*) extend to position +42 or +10 of topAI (wild-type +42, plasmid plasmid pGB217; wild-type +10, plasmid pGB215; rut* +42, plasmid pGB306; rut* +10, plasmid pGB305). Error bars represent ±1 standard deviation from the mean (n = 3).

topAI expression is induced in response to translation stress.

(A) Schematic representation of topAI-lux translational reporter fusions, indicating mutations in the start codon and Shine-Dalgarno sequence of the toiL uORF. (B) Luciferase activity of the wild-type topAI-lux translational reporter fusion (pGB202) in ΔtopAI-yjhQ cells (strain GB001) expressing either wild-type 23S rRNA (plasmid pGB322) or ΔT1917 23S rRNA (plasmid pGB318) in trans. Activity was measured four hours post-induction with propionate. (C) Luciferase activity of wild-type and mutant topAI-lux translational reporter fusions in ΔtopAI-yjhQ cells (strain GB001) grown in the presence of ribosome-targeting antibiotics. Cells were grown in LB medium to an OD600 of ∼1.0. Indicated samples were treated with tetracycline (0.5 μg/ml), spectinomycin (90 μg/ml), retapamulin (4 μg/ml), erythromycin (100 μg/ml), tylosin (400 μg/ml), or chloramphenicol (1 μg/ml). Luminescence was measured 90 min after antibiotic treatment. Dark gray bars show data for the wild-type topAI-lux translational reporter fusion (plasmid pGB202); light gray bars show data for the topAI-lux translational reporter fusion with a mutated toiL start codon (plasmid pGB313); white bars show data for the topAI-lux translational reporter fusion with a mutated toiL Shine-Dalgarno sequence (pGB366). Error bars represent ±1 standard deviation from the mean (n = 3).

Tetracycline treatment reduces premature Rho-dependent transcription termination within topAI-yjhQ and increases abundance of the associated mRNA.

(A) Schematic of the rhoL-rho and topAI-yjhQP operons showing the positions of PCR amplicons used for ChIP-qPCR and/or qRT-PCR. (B) Occupancy of RNAP measured by ChIP-qPCR in the rho gene. Occupancy values are normalized to those in rhoL (control PCR amplicon “C” in panel A). Data are shown for wild-type (strain MG1655) or rho mutant (R66S amino acid substitution in Rho; strain CRB016) cells grown in the presence or absence of tetracycline, as indicated. The x-axis label corresponds to the PCR amplicon shown in panel A. (C) Occupancy of RNAP measured by ChIP-qPCR at positions across the topAI-yjhQ transcript. Occupancy values are normalized to those in the topAI upstream region (control PCR amplicon “C” in panel A). (D) RNA levels measured by qRT-PCR at positions across the topAI-yjhQ transcript, normalized to the mreB gene. Error bars represent ±1 standard deviation from the mean (n = 3). Statistical significance is indicated as follows: n.s. = not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001.

A uORF, toiL, is located within the topAI 5’ upstream region.

(A) Schematic representation of toiL-lacZ translational reporter fusions, indicating mutations upstream and within the toiL uORF. (B) β-galactosidase activity of the wild-type toiL-lacZ translational reporter fusion (wt; plasmid pGB164), and fusions with mutations immediately upstream of toiL (TTG→tga; plasmid pGB201), at the toiL start codon (ATG→tga; plasmid pGB200), second codon (CTG→tga; plasmid pGB197), or fourth codon (AAT→tga; plasmid pGB196), in wild-type cells (strain MG1655). Error bars represent ±1 standard deviation from the mean (n = 3).

Structural changes in the topAI 5′ upstream region induced by tetracycline treatment.

(A) Predicted RNA secondary structure of the topAI 5’ UR, indicating in-cell SHAPE reactivities (ρ) of each base from an untreated sample. Values of ρ are indicated on a yellow-to-green gradient; white circles indicate no detected reactivity. The position of the toiL uORF is indicated by a red arrow, and the position of the start of topAI is indicated by a gray rectangle. Numbers indicate position relative to the mRNA 5’ end. (B) Filled red or green circles indicate bases with significant changes in SHAPE reactivity when cells were treated with tetracycline (p < 0.05).

Tetracycline stalls ribosomes on start codons.

(A) Normalized Ribo-seq coverage for all annotated ORFs for cells (strain MG1655) grown ± tetracycline. (B) Heatmap showing normalized Ribo-seq coverage in the region around the toiL uORF in untreated cells (no antibiotic) or tetracycline-treated cells. The color indicates the sequence-read coverage for RNA fragment 3’ ends that are presumed to represent the downstream edge of ribosome footprints. The toiL uORF position and encoded amino acid sequence is indicated. (C) Average of normalized Ribo-seq coverage for the regions around start codons for all annotated ORFs, for untreated cells (gray line; no antibiotic) or tetracycline-treated cells (orange line). Sequence-read coverage was calculated for RNA fragment 3’ ends that are presumed to represent the downstream edge of ribosome footprints.

Ribosome stalling at the topAI leader in vivo.

(A) Schematic showing the toiL-ermCL-lux stalling reporter in the translationally inactive conformation (“OFF”) that is expected in the absence of ribosome stalling, and the translationally active conformation (“ON”) that is expected when ribosomes stall within toiL sequence close to the junction of toiL with ermCL. (B) Schematic showing toiL-ermCL stalling reporters where toiL is progressively extended by one codon at the 3’ end and fused to part of ermCL. toiL sequence is indicated by red arrows; ermCL sequence is indicated by green arrows. The last indicated stalling reporter extends to position 5 of toiL but has a Val5→Leu substitution (blue). (C) Luciferase activity of the toiL-ermCL-lux stalling reporters (plasmids pGB323, pGB346, pGB325, pGB326, pGB324, pGB328, pGB329, pGB307, pGB347, and pGB327) in ΔtopAI-yjhQ cells (strain GB001). Cells were grown to an OD600 of ∼1.0 and treated with the indicated ribosome-targeting antibiotics at the same concentrations as in Figure 2C. Luminescence was measured three hours post-treatment. Horizontal dashed lines indicate luminescence activity of 1, to facilitate comparison between panels with different y-axis scales. Error bars represent ±1 standard deviation from the mean (n = 3).

Model for regulation of topAI expression.

Schematic showing a model for regulation of topAI expression. In the repressed state, a hairpin forms between the toiL sequence and the ribosome-binding site of topAI, repressing translation, which in turn promotes premature Rho-dependent transcription termination within the topAI gene. When cells are treated with certain ribosome-targeting antibiotics such as tetracycline, ribosomes stall within toiL in a sequence-specific manner, preventing formation of the repressive hairpin.