Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate

Abstract

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins.

Figure 1.

A small non-coding RNA, IsrA, co-purifies with RNA polymerase. (A) Pull-down of RNAs with 6xHis-tagged (strain RL^rpoCHIS) or tag-less RNAP on Ni²⁺-NTA sepharose. Isolated RNAs were 5′- or 3′ end labeled and analyzed as described in the Materials and Methods. (B) Secondary structure model of the purified RNA, identified as IsrA ((23); a.k.a. IS61 (20) or McaS (21,22)) which was based on phylogenetic analysis (see Supplementary Figure S2). An extension that substitutes the outer portion of stem 3 and provides affinity to streptomycin (streptotag (32); see Figure 2) is boxed. (C) Summary of IsrA structure in sequence logo representation; height of nucleotides mark the degree of conservation. Base pairs that form stems are indicated by brackets; rectangles indicate regions implicated in protein binding (CsrA), or translational regulation of flhDC, or csgD mRNAs (21,22,43). (D) Purification of RNAP on streptavidin sepharose through a biotinylated tag at the β’ subunit that can be cleaved off using HRV3C (3C) protease. RNAPs were isolated from strains; lane 1: RLΔisrA-pisrA (tag-less RNAP, IsrA under mutant promoter on multi-copy plasmid); lane 2: RL (tag-less RNAP, genomic IsrA under mutant promoter); lane 3: MG1655^rpoCBCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 4: JC7623^rpoCBCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 5: RL^rpoCBCCP (BCCP-tagged RNAP, genomic IsrA under mutant promoter); lane 6: RL^rpoCBCCPhfq65 (BCCP-tagged RNAP, genomic IsrA under mutant promoter, Hfq lacks C-terminus) (see also Supplementary Table). Relative expression levels of IsrA are shown above the gels (expressed from wild-type promoter in chromosomal locus (+), expressed from a mutant promoter in the chromosomal locus (++) or from mutant promoter from a high-copy plasmid (+++)). RNAPs were released by HRV3C (3C) cleavage and analyzed by SDS/PAGE on a 4–20% gradient gel. Strains that have RNAP without a biotinylated tag were used as a control. Lanes 7 and 8 are controls for non-specific binding to and efficiency of release from beads of RNAP in lanes 1 and 5, respectively. (E) Northern blot analysis of RNAs that co-purified with biotinylated RNAP in panel (D). The blots were probed against IsrA (bottom) or 6S RNA (top). Indicated are the presence of the biotinylated affinity tag on RNAP β’ (+ or – tag), the expression levels of IsrA in the input extract (as in panel (D)), and whether Hfq in the cells was truncated to its core of 65 amino acids (ΔC) or was wild type (+).

Figure 2.

RNAP and a defined set of proteins co-purify with IsrA carrying a streptotag. (A) Streptotagged IsrA-3tag (see Figure 1B) and proteins associated with it were isolated on di-streptomycin beads from strain in which the endogenous gene for IsrA was disrupted (strain RL^rpoCBCCP ΔisrA-pisrA3tag, see Supplementary table). Co-purifying proteins (Strepto lanes) were analyzed by SDS-PAGE on 4–20% gradient gels and stained with Coomassie or silver as described in the Materials and Methods. Mock isolation with the di-streptomycin beads using the parental strain with untagged IsrA (RL; lane 4) was used as a negative control. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively. RNAP purified on a heparin column from strain BW-62 was used as size control (RC; lane 3). (B) RNA was analyzed by Northern blotting with an IsrA-specific probe. Note that the blot is overexposed to make the IsrA band in the input visible. The presence and absence of affinity tags on IsrA is indicated by + and –, respectively.

Figure 3.

IsrA specifically binds core enzyme of RNAP. (A) Electrophoretic mobity shift assay (EMSA) with radioactively labeled, gel-purified T7 synthesized ³²P-labelled RNAs (tRNA^Ala, 6S RNA or IsrA) which were mixed with equimolar amounts of purified core or holo (σ⁷⁰) enzymes of RNAP. The labeled RNA and a competitor (T7A1 promoter DNA or heparin) were added to RNAP first (+*) or second (+). (B) Competition of various unlabeled RNAs with ³²P-labeled 6S RNA and IsrA. 15-fold excess of unlabeled competitor RNA (Supplementary Figure S5) was added to RNAP before addition of labeled RNAs. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added to all reactions before loading on the gel to dissolve non-specific complexes.

Figure 4.

Stem 2 of IsrA is required for specific binding to RNAP. (A) Mutant versions of IsrA (see also Supplementary Figure S1F) shown as a black line-diagram with deleted regions indicated in white. In the i2loop mutant the loop closing stem 2 has been replaced with GAAG (gray; see also Supplementary Figure S6A). (B) EMSA of RNAP and mutant labeled IsrA RNAs with heparin added before (+*) or after (+) complex formation. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. (C) Competition of unlabeled mutant IsrA RNAs (15-fold molar excess) with labeled wild-type variant. Unlabeled competitor RNA was always added before complex formation of RNAP with labeled RNA. All reactions contained total yeast RNA as a non-specific competitor prior to complex formation. Heparin was added in all reactions after formation of complexes. Only IsrA mutants that are able to bind RNAP in the presence of heparin (see panel B) compete against association of labeled IsrA. (D) Northern blot analysis of mutant IsrA RNAs that co-purified with RNAP (for protein gel see Supplementary Figure S6B. Plasmids from which the mutant IsrA RNAs (see panel A) were expressed, along with an empty vector (pGemT), were transformed into strain RL^rpoCBCCPΔisrA with a disrupted IsrA gene and carrying biotinylated RNAP. A strain expressing RNAP without the biotinylated tag (RLΔisrA-pisrA(*)) was used as a negative control (input and pull-down are overloaded). sRNAs were released from beads with 3C, and the blots were probed against IsrA (bottom) or 6S RNA (top). Below the blot, quantitation of the bands corresponding to IsrA mutants (encircled in the blot), normalized to the corresponding bands of 6S RNA.

Figure 5.

Only non-specific binding of IsrA to RNAP can inhibit transcription. (A) Transcription on a linear template containing the T7A1 promoter. The order in which RNAP was allowed to associate with the promoter DNA or competitor RNA is given (+* added first; + second). Note the template-independent labeling of IsrA at the 3′ end by RNAP, which suggests non-specific binding near the active center of RNAP (see text). (B) Pre-formed 11-mer elongation complexes were chased in the presence or absence of sRNAs. (C) Abortive synthesis of CpApU on T7A1 promoter. Mutant IsrA RNAs or heparin were added before promoter DNA. Note that the IsrA variant Δ2, which cannot bind RNAP specifically, still inhibits initiation and is labelled at the 3′ end (see text for details).

Figure 6.

Hypothetical model of co-transcriptional functions of sRNPs formed by IsrA, and possibly other sRNAs. (A) Transcription regulation. IsrA stabilized by protein(s) such as Hfq and/or S1 binds specifically to core enzyme of RNAP via its stem 2, with or without the release of the proteins. During transcription elongation IsrA may be involved in regulation of transcription through interactions with nucleic acids of the elongation complex or by changing RNAP response to regulatory signals. (B) Translation regulation (note that shown is co-transcriptional regulation of translation, though our data do not exclude regulation that is independent of RNAP). sRNA associates with one or a set of proteins form sRNPs as directed by cellular requirements. Protein(s) of the sRNP remain silent until they are delivered to the target mRNA. The sRNP can associate with elongating RNAP (via stem 2 of IsrA), as suggested by our data, which would make its delivery to the target mRNA more efficient. Target mRNA is recognized via limited complementarity with the sRNA. Recognition of the target facilitates delivery of the protein(s) of the sRNP, which, depending on the set of proteins, determine the fate of the mRNA, such as activation or inhibition of translation or degradation of the mRNA. The sRNA can participate in the regulation of the mRNA along with the proteins or is released.