Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs

Abstract

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.

Keywords: Hfq; RNA hairpin; Rho-independent terminator; bacterial sRNA; premature termination.

FIGURE 1.

Effect of extension of the terminator stem of sgrS on transcription termination. (A) DNA sequences of sgrS-S-rplLT and sgrS-S-LS1-rplLT. The sequence corresponding to the sgrS-S is shown as regular letters, whereas the terminator sequence derived from the rplL is shown as italic letters. The inverted repeat sequences are indicated by horizontal arrows. Nucleotides are numbered from the site corresponding to the 5′ end of sgrS-S. The inserted sequences to stabilize the terminator stem are shown as bold letters. (B) The predicted secondary structures and the thermodynamic stabilities (ΔG, kcal/mol) of terminator RNA hairpins without poly(U) sequence were determined according to the Mfold program (Zuker 2003). (C) Analysis of transcription termination. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A₆₀₀ = 0.6, 0.02% arabinose was added and incubation was continued for 5 min. Total RNAs were prepared, and 10 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively.

FIGURE 2.

The effect of extension of the terminator stem of sgrS-S on the generation of heterogeneous shorter transcripts. (A) DNA sequences of sgrS-S and its variants. The inverted repeat sequences of the terminator are indicated by horizontal arrows. The inserted sequences to stabilize the terminator stem are shown as bold letters in sgrS-S-LS1 and sgrS-S-LS2. The sequences derived from the rrnBT and rplLT are shown as bold letters in sgrS-S-LS3 and sgrS-S-LS4, respectively. Nucleotides are numbered from the site corresponding to the 5′ end of sgrS-S. (B) The predicted secondary structures and the thermodynamic stabilities (ΔG, kcal/mol) of terminator RNA hairpins without a poly(U) sequence were determined according to the Mfold program (Zuker 2003). (C) The effect of extension of the terminator stem of sgrS-S on the generation of shorter transcripts. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A₆₀₀ = 0.6, 0.2% arabinose was added and incubation was continued for 10 min. Total RNAs were prepared, and 20 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts. (D) The effect of extension of the terminator stem of sgrS-S on the generation of shorter transcripts under glucose-phosphate stress. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A₆₀₀ = 0.6, 0.01% αMG was added and incubation was continued for 10 min. Then, 0.2% arabinose was added and incubation was continued for 10 min. Total RNAs were prepared, and 20 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts. (E) The effect of replacement of the terminator hairpin of sgrS-S with those derived from rrnBT and rplLT on the generation of shorter transcripts. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium and treated as described in D. Total RNAs were prepared, and 20 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts.

FIGURE 3.

Properties of heterogeneous transcripts. (A) The effect of hfq backgrounds on the expression of heterogeneous transcripts. TM542 (ΔsgrS) and TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A₆₀₀ = 0.6, 0.1% αMG was added and incubation was continued for 10 min, and then 0.02% arabinose was added and incubation was continued for 5 min. Total RNAs were prepared, and 10 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts. (B) An examination of Hfq binding of heterogeneous transcripts. TM803 (ΔsgrS hfq-Flag) cells harboring pSgrS-S-LS2 were grown in LB medium. At A₆₀₀ = 0.6, 0.1% αMG was added and incubation was continued for 10 min, and then 0.02% arabinose was added and incubation was continued for 5 min. Crude extract was prepared and subjected to the pull-down assay using anti-Flag agarose as described in Materials and Methods. (C,D) The effect of extension of the terminator stem of sgrS-S on SgrS function in the presence (D) and absence (C) of glucose-phosphate stress. TM542 (ΔsgrS) cells harboring indicated plasmids were grown in LB medium. To prepare total RNAs from cells without the stress, 0.02% arabinose was added at A₆₀₀ = 0.6 and incubation was continued for 5 min (C). To prepare total RNAs from cells with the stress, the culture of A₆₀₀ = 0.6 was exposed to 0.1% αMG for 10 min, and then 0.02% arabinose for 5 min (D). Ten micrograms of RNA samples was subjected to Northern blotting using the ptsG probe.

FIGURE 4.

Analysis of the 3′ ends of transcripts derived from sgrS-S and sgrS-S-LS2. TM542 (ΔsgrS) cells harboring pSgrS-S (A) or pSgrS-S-LS2 (C) were grown in LB medium. At A₆₀₀ = 0.6, 0.1% αMG was added and incubation was continued for 10 min, and then 0.02% arabinose was added and incubation was continued for 5 min. Total RNAs were prepared and duplicate RNA samples (10 µg) were resolved side-by-side on a 12% polyacrylamide gel electrophoresis in the presence of 8 M urea. One side of the gel was subjected to Northern blotting (A,C). The region corresponding to transcripts was cut out from the other side of the gel (A,C). A single gel piece containing the SgrS-S band was used for transcripts from sgrS-S (A). The gel was divided into three gel pieces (upper, middle, and lower) for transcripts from sgrS-S-LS2 (C). The upper gel piece corresponds to the full-length transcript, whereas the middle and lower gel pieces correspond to heterogeneous shorter transcripts. RNAs were purified from the gel pieces and subjected to 3′-RACE analysis. DNA sequences corresponding to the 3′ region of transcripts were determined by using randomly picked-up plasmid clones containing amplified cDNAs. The sequences and number of clones analyzed are shown (B,D). Others represent the clones in which the 3′ ends of RNAs were mapped in the region prior to the terminator T residue stretch (D).

FIGURE 5.

(A) Effect of mutation in 3′ exoribonuclease genes on generation of shorter transcripts. TM542 (ΔsgrS), TM894 (ΔsgrS Δpnp), TM895 (ΔsgrS Δrnb), and TM896 (ΔsgrS Δrnr) cells harboring pSgrS-S-LS2 were grown in LB medium containing 0.2% arabinose. At A₆₀₀ = 0.6, total RNAs were prepared and 10 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe. (B,C) Stability of heterogeneous transcripts in the presence (C) and absence (B) of stress. TM772 (ΔsgrS Δhfq) cells harboring pSgrS-S-LS2 were grown in LB medium. For the preparation of samples in the absence of stress, 0.2% arabinose was added at A₆₀₀ = 0.6 and incubation was continued for 10 min, and then rifampicin (250 µg/mL) was added. Total RNAs were prepared at the indicated time after the addition of rifampicin. For the preparation of samples in the presence of stress, cells were exposed to 0.01% αMG for 10 min prior to the addition of arabinose. The RNA samples (20 µg) were subjected to Northern blotting using the SgrS-S. Arrowheads represent the full-length transcripts.

FIGURE 6.

Analysis of transcription of sgrS-S and sgrS-S-LS2 directed by the tac promoter. (A) DNA sequences of the chimeric P_tac-sgrS-S and P_tac-sgrS-S-LS2 genes. The sequence corresponding to sgrS-S is shown as regular letters, whereas the tac promoter sequence is shown as italic letters. The sequences of the −10 and −35 regions of the tac promoter are boxed. The inverted repeat sequences of the sgrS-S terminator are indicated by horizontal arrows. The inserted sequences to stabilize the terminator stem are shown as bold letters. (B) In vivo expression of the P_tac-sgrS-S and P_tac-sgrS-S-LS2. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. Total RNAs were prepared at A₆₀₀ = 0.4, and 20 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe. Arrowheads represent the full-length transcripts. (C) SDS-PAGE analysis of the purified RNAP-Flag. The affinity-purified PNAP-Flag sample (5 µL) was analyzed by SDS-PAGE and Coomassie staining as described in Materials and Methods. The bands corresponding to RNAP subunits are indicated on the left. Protein size markers are shown on the right. (D) In vitro transcription of the P_tac-sgrS-S and P_tac-sgrS-S-LS2. The transcription reaction was performed as described in Materials and Methods. The samples were subjected to Northern blotting using the SgrS-S probe. Arrowheads represent the full-length transcripts.

FIGURE 7.

The effect of extension of the terminator stem of ryhB on transcription termination and on properties of RyhB. (A) DNA sequences of ryhB and its variants. The inverted repeat sequences of terminators are indicated by horizontal arrows. The inserted sequences to stabilize the terminator stem are shown as bold letters. Nucleotides are numbered from the site corresponding to the 5′ end of ryhB. (B) The predicted secondary structures and the thermodynamic stabilities (ΔG, kcal/mol) of RNA hairpins without the poly(U) sequence were determined according to the Mfold program (Zuker 2003). (C) Generation of heterogeneous transcripts and effect of hfq backgrounds. TM635 (ΔryhB) and TM820 (ΔryhB Δhfq) cells harboring the indicated plasmid were grown in LB medium. At A₆₀₀ = 0.6, 250 µM of 2, 2′-dipyridyl was added and incubation was continued for 10 min. Then, 0.02% arabinose was added and incubation was continued for 5 min. Total RNAs were prepared, and 1.6 or 0.25 µg of RNA samples was subjected to Northern blotting using the RyhB probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts. (D) Effect of extension of the terminator stem on RyhB function. Two micrograms of RNA samples described in Figure 7C was subjected to Northern blotting using the sodB probe.