Journal: Nucleic Acids Research

Article Title: 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

doi: 10.1093/nar/gkv1302

Figure Lengend Snippet: A small non-coding RNA, IsrA, co-purifies with RNA polymerase. ( A ) Pull-down of RNAs with 6xHis-tagged (strain RL rpoC HIS ) or tag-less RNAP on Ni 2+ -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 (; a.k.a. IS61 or McaS ( , )) 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 ; see Figure ) 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 ( , , ). ( 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: RLΔ isrA -p isrA (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 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 4: JC7623 rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under wild-type promoter); lane 5: RL rpoC BCCP (BCCP-tagged RNAP, genomic IsrA under mutant promoter); lane 6: RL rpoC BCCP hfq65 (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 (+).

Article Snippet: Cell-debris was removed by centrifugation (2 × 10 min 7K rpm, 20 min 18K rpm) and the lysates were filtered over Millex-HV 0.45 μm PVDF filter-units (Millipore) before mixing with Ni Sepharose 6 Fast Flow or Streptavidin Sepharose High performance (GE Healthcare) beads.

Techniques: Isolation, Labeling, Purification, Sequencing, Protein Binding, Mutagenesis, Plasmid Preparation, Expressing, SDS Page, Binding Assay, Northern Blot