A role for Rho-dependent polarity in gene regulation by a noncoding small RNA

Abstract

Gene regulation by bacterial trans-encoded small RNAs (sRNAs) is generally regarded as a post-transcriptional process bearing exclusively on the translation and/or the stability of target messenger RNA (mRNA). The work presented here revealed the existence of a transcriptional component in the regulation of a bicistronic operon-the chiPQ locus-by the ChiX sRNA in Salmonella. By studying the mechanism by which ChiX, upon pairing near the 5' end of the transcript, represses the distal gene in the operon, we discovered that the action of the sRNA induces Rho-dependent transcription termination within the chiP cistron. Apparently, by inhibiting chiP mRNA translation cotranscriptionally, ChiX uncouples translation from transcription, causing the nascent mRNA to become susceptible to Rho action. A Rho utilization (rut) site was identified in vivo through mutational analysis, and the termination pattern was characterized in vitro with a purified system. Remarkably, Rho activity at this site was found to be completely dependent on the function of the NusG protein both in vivo and in vitro. The recognition that trans-encoded sRNA act cotranscriptionally unveils a hitherto neglected aspect of sRNA function in bacteria.

Figure 1.

(A) Effect of chiX and rho mutations on chiQ-lacZY expression. ChiX sRNA down-regulates expression of a chiQ-lacZY fusion by pairing with a sequence upstream of the chiP gene (Figueroa-Bossi et al. 2009). Mutating the chiP initiation codon (from AUG to AGG) silences chiQ-lacZY expression regardless of the chiX allele (A) in spite of the fact that the intercistronic region between chiP and chiQ (B) contains a Shine-Dalgarno motif for independent chiQ translation (overlined in B). As a result, a strain carrying chiP_AGG chiQ-lacZY is unable to grow on lactose as the sole carbon source (Lac⁻ phenotype). (C) Mutations in the rho gene relieve chiQ-lacZY silencing to various degrees (middle) and restore growth on lactose (left). (Right) A similar effect is obtained by exposing cells to Rho inhibitor Bcm. In the latter test, wild-type rho bacteria carrying chiP_AGG chiQ-lacZY were spread onto a minimal lactose plate, and a filter paper disc wet with 3 μL of a 50 mg/mL Bicyclomycin (Bcm) solution was applied in the center of the plate. Diffusion of the drug generated a concentration gradient. After a 2-d incubation, a ring of growth was observed in the region of the plate where the Bcm concentration was low enough not to inhibit growth but sufficiently high to interfere with Rho activity. Strains used were MA10608 (rho^wt), MA10633 (rho G63D), MA10634 (rho K130Q), and MA10769 (rho Y80C). For the complete genotypes, see Supplemental Table S1.

Figure 2.

ChiX sRNA-induced transcriptional polarity and its relief by rho mutations. (A) Effect of rho G63D on the expression of a chiQ-lacZ fusion. Stimulation of lacZ expression by rho G63D requires a functional chiP promoter and is independent of sequences in the intercistronic region between chiP and chiQ. Strains used were (from left to right) MA9655, MA9860, MA10235, MA10240, MA10277, and MA10295. (B) chiPQ transcription profile in strains carrying temperature-sensitive RNase E allele rne-3071 (Figueroa-Bossi et al. 2009). Exponentially growing cells were incubated for 15 min at 43°C prior to RNA extraction. RNA was separated on a 1.3% agarose-formaldehyde gel and probed with a ³²P-labeled DNA oligonucleotide complementary to a sequence near the 5′ end of chiPQ mRNA (pp977; “probe 1” in the diagram). Strains used were (from left to right) MA9826, MA9816, and MA9886. (C) Transcription in the distal portion of the chiPQ operon. RNA turnover products from the 3′ end of the chiPQ mRNA are quantified by Northern blot hybridization. RNA was extracted from exponentially growing cultures of strains MA3409 (chiP^wt, rho^wt), MA9870 (chiP^wt, rho G63D), MA10274 (chiPΔ22, rho^wt), and MA10293 (chiPΔ22, rho G63D); fractionated in a polyacrylamide gel; and probed with a ³²P-labeled oligonucleotide complementary to a sequence near the 3′ end of the chiPQ transcript (ppA83; “probe 2” in the diagram). (D) Effect of rho mutations or Bcm treatment on the synthesis of a S. aureus tRNA inserted in the intercistronic spacer of chiPQ mRNA. Bcm was used at a final concentration of 10 μg/mL. Total RNA extracted from stationary cultures was processed for Northern blotting as in C. The blot was hybridized to an oligonucleotide complementary to tRNA^S.a (ppJ62). RNA bands were quantified by PhosphorImaging using the ImageQuant program; 5S was used for normalization. Strains used were MA3409 (wild-type: lacks tDNA^S.a), MA11115 (rho^wt), MA11150 (chiP^wt, rho G63D), MA11168 (chiP^wt, rho Y80C), and MA11169 (chiP^wt, rho K130Q) (left panel); and MA11123 (chiP^wt ΔchiX) and MA11115 (rho^wt) without and with Bcm (right panel).

Figure 3.

Polarity of tandem UAA codons in the chiP gene. The sequence UAAUAA was inserted in place of tandem sense codons at different positions of the chiP gene in the chromosome of a strain carrying chiQ-lacZ. (A) Effect of rho mutation on the polarity gradient. Loss of lacZ stimulation by rho G63D in the interval between UAA-63 and UAA-108 (from 5.6-fold to 2.3-fold) suggests the presence of a polarity site within this interval. Strains used were MA10802 (UAA-10), MA10960 (UAA-46), MA10961 (UAA-63), MA11046 (UAA-78), MA11047 (UAA-108), and MA10445 (UAA-256). (B) Nucleotide sequence of the 5′ portion of the chiPQ mRNA. The ChiX pairing sequence and chiP-initiating AUG are boxed in yellow and orange, respectively. Gray boxes denote the positions of the double UAA codons. An octameric rut site element and the sequences found at the 3′ ends of in vitro terminated transcripts (see the text) are boxed in green and red, respectively.

Figure 4.

Mutational analysis of the chiP rut site. An octameric CCUUUCUC sequence, in the polarity interval identified by the UAA scanning analysis (see the text), was randomly mutagenized as described in the text in a strain carrying chiQ-lacZ. Most of the clones obtained expressed lacZ at higher levels than the parent strain, consistent with the role of the C-rich octamer in the polarity effects. (A) Growth phenotype of representative mutants on a minimal (NCE) lactose plate. (B) Effects of rut site changes on chiQ-lacZ expression in wild-type and rho G63D mutant backgrounds. The strains used in this experiment are listed in Supplemental Table S1. (C) Sequence alignment of the chiP rut region from members of the Enterobacteriaceae family. Cytosine residues are highlighted in green.

Figure 5.

Effect of Rho and NusG on in vitro transcription of DNA templates containing the upstream section (ChiP1 template) or the entirety (ChiP2 template) of the chiP gene fused to the T7A1 promoter. Runoff (RO) transcripts are 560 and 1575 nt long for transcriptions with ChiP1 (A) and ChiP2 (B), respectively. Longer transcripts presumably result from RNA polymerase continuing transcription after reaching the end of a DNA template and “switching” to another template molecule (Nudler et al. 1996; Rabhi et al. 2011). The concentrations of Rho hexamers and Bcm were 70 nM and 150 μM, respectively. The concentration of NusG was 70 nM (+ lanes) or 140 nM (++ lanes).

Figure 6.

Mutations in nusG gene relieve chiP polarity. Mutants were obtained by combining random PCR mutagenesis and chromosomal recombineering as described in the text. (A) Effect of nusG mutations on the expression of chiQ-lacZ in a chiP_AGG mutant background. Strains used were MA11167 (nusG^wt), MA11158 (nusG 174fs), MA11159 (nusG F144I), MA11161 (nusG V162D), and MA11162 (nusG F141S). (B) Effect of nusG mutations on the synthesis of S. aureus tRNA (see the legend for Fig. 2D). Total RNA extracted from stationary cultures was processed for Northern blotting as in Figure 2D. The blot was hybridized to an oligonucleotide complementary to tRNA^S.a (ppJ62). Strains used were MA11170 (nusG^wt), MA11171 (nusG 174fs), MA11172 (nusG 144I), and MA11173 (nusG V162D).