Volume 9, Issue 1, Pages 23-30 (January 2002) Hfq  Thorleif Møller, Thomas Franch, Peter Højrup, Douglas R Keene, Hans Peter Bächinger, Richard G Brennan, Poul Valentin-Hansen  Molecular Cell  Volume 9, Issue 1, Pages 23-30 (January 2002) DOI: 10.1016/S1097-2765(01)00436-1

Figure 1 Hfq Is Essential for Spot 42 RNA Stability Exponential grown cultures of strains NU426 (hfq+), TX2822 (hfq1), and TX2761 (hfq2) were treated with rifampicin to block new transcription. Samples were taken at the indicated times and total RNA was extracted. Spot 42 RNA levels were analyzed by Northern blot analysis. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 2 Hfq Forms a Complex with Spot 42 RNA (A) Coimmunoprecipitation of Hfq and Spot 42 RNA. Cell extracts of strains NU426 (hfq+) and TX2822 (hfq1) were incubated with Hfq-specific antisera, and immunoprecipitated complexes were bound to protein A agarose. Total immunoprecipitated RNA was extracted and examined for the presence of Spot 42 RNA and 6S RNA by Northern blot analysis. Lanes 1 and 4 (fraction I, control), total RNA from cells extracts; lanes 2 and 5 (fraction II, control), RNA extracted from protein A agarose of untreated extracts; lanes 3 and 6 (fraction III), RNA extracted from protein A agarose of extracts incubated with antisera. Mature 6S as well as a precursor 6S with a 5′ end extension are detected in fraction I. (B) Gel mobility shift assays of Hfq binding to Spot 42 RNA. Samples containing 5′ end-labeled transcript of Spot 42 RNA (4 nM final concentration) and a 500-fold molar excess of tRNA were incubated with increasing amounts of Hfq, in the absence (lanes 1–4) or in the presence of unlabeled Spot 42 RNA (lanes 5–10). Final monomer-concentrations of Hfq were 20 nM (lanes 2, 5, and 8), 100 nM (lanes 3, 6, and 9), and 500 nM (lanes 4, 7, and 10). No protein was added to the binding reactions (lanes 1, 5, and 8). Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 3 Hfq Binds to Single-Stranded U-Rich Sequences of Spot 42 RNA (A) Hydroxyl radical footprints of free and Hfq-bound Spot 42 RNA. Samples containing 5′ end-labeled transcript of Spot 42 RNA (5 nM final concentration) were incubated with increasing amounts of Hfq. No protein added, lane 3; 5, 50, 500, and 1500 nM of Hfq were present in the binding reactions, lanes 4–7, respectively. Alkaline hydrolysis ladder, lane 1. RNase T1 cleavage of Spot 42 RNA under denaturing condition (G-specific cleavage), lane 2. Bars mark regions of Spot 42 RNA that are strongly protected in the presence of Hfq. (B) Secondary structure of Spot 42 RNA (T. Møller et al., submitted). Arrows indicate the Sm-like sites protected by Hfq. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 4 The Sm1 Sequence Motif Is Conserved in Hfq Proteins Amino acid sequence alignment of a subset of bacterial Hfq proteins. The Sm consensus based on 80 Sm and Sm-related proteins (Achsel et al., 1999) is shown at the bottom together with the amino acid sequence and secondary structure of the Archaeoglobus fulgidus Sm1 protein (residues forming the uracil binding pocket are labeled “#”) (Törö et al., 2001). The Sm1 sequence motif of Hfq proteins is highlighted in gray. Positions that are identical in most Sm motifs are shown in bold in the Sm consensus. “h” signifies a bulky hydrophobic residue (L, I, M, V, F, Y, or W), and “∼” signifies segment of variable sequence and length. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 5 Hfq Forms a Doughnut-Shaped Structure Rotary shadowed electron micrograph of purified E. coli Hfq. Note, rotary-shadowed particles are covered with carbon and platinum, and therefore such particles appear larger than negatively stained particles. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 6 Hfq Cooperates in RNA-RNA Interaction (A) Incubations containing 5′ end-labeled Spot 42 RNA (5 nM), 2 μM tRNA, and increasing amounts of unlabeled galK′ substrate were incubated in the absence (lanes 2–4) or in the presence of Hfq (lanes 6–8), and complex formation was monitored in a electrophoretic mobility shift experiment. galK′ RNA concentrations were 20 nM (lanes 2 and 6), 100 nM (lanes 3 and 7), and 500 nM (lanes 4 and 8), and the Hfq “monomer” concentration was 1 μM. Unbound Spot 42 RNA (I) and complexes corresponding to Hfq-Spot 42 RNA (II), Spot 42 RNA-galK′ RNA (III), and Spot 42 RNA-Hfq-galK′ RNA (IV) are indicated. (B) Nuclease probing of Spot 42-RNA-galK′-RNA and Spot 42-RNA-Hfq-galK′-RNA complexes. Incubations containing 5′ end-labeled Spot 42 RNA (5 nM), 3 μM tRNA, and increasing amounts of unlabeled galK′ substrate were incubated in the absence (lanes 1–5) or in the presence of Hfq (lanes 6–10) and treated with RNase T2. Lanes marked T1, OH−, and P are RNase T1 cleavage of Spot 42 RNA under denaturing condition (G-specific cleavage), alkaline hydrolysis ladder of Spot 42 RNA, and untreated Spot 42 RNA probe, respectively. The galK′ RNA concentrations for lanes 1–5 were 0, 100, 500, 1000, and 2000 nM, respectively, and for lanes 6–10 were 0, 4, 20, 100, and 500 nM, respectively. The Hfq “monomer” concentration was 1 μM (lanes 6–10). Arrows indicate reduced cleavage. Black bar indicates nucleotides that are inaccessible or only weakly accessible to T2 cleavage in the presence of Hfq. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)

Figure 7 Protein-Mediated RNA-RNA Interaction (A) Secondary structure of Spot 42 RNA. The galK complementary regions involved in base pairing are shown in a gray background. (B) Sequence of the translational initiation region of galK. The Spot 42 RNA complementary regions involved in duplex formation (Figure 6B) (T. Møller et al., submitted) are shown in a gray background, and Sm-like sites are indicated in bold. The galT stop codon and the galK start codon are underlined. Molecular Cell 2002 9, 23-30DOI: (10.1016/S1097-2765(01)00436-1)