Journal: eLife

Article Title: Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

doi: 10.7554/eLife.40576

Figure Lengend Snippet: ( A ) Schematic of CoSMoS transcription initiation rate measurement in the presence of GreB or DksA. DNA molecules (314 bp) containing the rrnB P1 promoter (bent arrow) and labeled with AF488 (blue star) were tethered to the chamber surface via a biotin-neutravidin linkage. Initiation in the presence or absence of DksA, GreB, and/or ppGpp is detected as co-localization of two oligonucleotide probes labeled with TAMRA (green star) that hybridize near the 5′ end of the nascent transcript. ( B ) Example transcript probe fluorescence images (1.1 × 1.1 µm) taken at 5 s intervals (top) and corresponding intensity record (bottom) from the location of a single template DNA molecule. An objective image analysis algorithm (see Materials and methods) detected a spot of probe fluorescence at the times shown in green. ( C–F ) Cumulative distributions of the time until first probe detection on each DNA molecule. Only probe colocalization events 5 s or longer in duration were scored. Measurements were made in the absence (purple) or presence (green and blue) of secondary channel factors GreB ( C, E ) or DksA ( D, F ), without ( C, D ) or with ( E, F ) 100 μM ppGpp. Exponential fits (lines) are corrected (see Materials and methods) for non-specific binding of probe to the chamber surface measured at control locations that lack DNA molecules (gray). ( G ) Inhibition of initiation by secondary channel factors with and without 10 nM GreB, 100 nM DksA, and/or 100 μM ppGpp derived from experiments in ( C–F ), taken from the fit parameters . Graph indicates the rate of initiation in the presence of the indicated factors (blue and green) relative to that measured in the same experiment without the factors (purple). Error bars: S.E.

Article Snippet: The template was synthesized by PCR using AF488- and biotin-labeled primers 5’-/AF488/GCGGTCAGAAAATTATTTTAAATTTCC-3’ and 5’-/5Biosg/CGTGTTCACTCTTGAGACTTGGTATTC-3’ (IDT DNA), E. coli genomic DNA from strain MG1655 (ATCC), and Herculase II Fusion DNA polymerase (Agilent) according to the manufacturer’s protocol.

Techniques: Labeling, Fluorescence, Binding Assay, Control, Inhibition, Derivative Assay

Journal: eLife

Article Title: Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

doi: 10.7554/eLife.40576

Figure Lengend Snippet: ( A ) Experiment design. Template and labeled proteins were the same as in . DNA was first imaged, then the AF488 dye (blue star) was bleached (white star). Then AF488 oligonucleotide probes and 1 mM NTPs were introduced as in , along with 1 nM σ 70 RNAP 647 and 10 nM GreB Cy3B . ( B ) Two excerpts from the record of fluorescence intensities colocalized with one template DNA molecule. Two events in which σ 70 RNAP 647 bound to DNA accompanied by GreB Cy3B (orange) and one in which σ 70 RNAP 647 arrived alone (purple) are highlighted. Only the last resulted in transcription initiation as judged by AF488-probe colocalization. Time series images (1.1 × 1.1 µm) from the time interval delimited by dashed lines confirm appearance of co-localized AF488-probe and σ 70 RNAP 647 .

Article Snippet: The template was synthesized by PCR using AF488- and biotin-labeled primers 5’-/AF488/GCGGTCAGAAAATTATTTTAAATTTCC-3’ and 5’-/5Biosg/CGTGTTCACTCTTGAGACTTGGTATTC-3’ (IDT DNA), E. coli genomic DNA from strain MG1655 (ATCC), and Herculase II Fusion DNA polymerase (Agilent) according to the manufacturer’s protocol.

Techniques: Labeling, Fluorescence

Journal: Molecular Cell

Article Title: A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation

doi: 10.1016/j.molcel.2021.05.032

Figure Lengend Snippet: The KH-S1 portal is crucial for PNPase-sRNA-Hfq complex formation (A–C) Electrophoretic mobility shift assays (EMSAs) of wild-type PNPase and KH-S1 mutants with 400 nM RyhB (A), 3ʹETS leuZ (B), and CyaR (C) in the absence and presence of 400 nM Hfq hexamer. Ternary complexes are highlighted with a red dot. Two different PNPase concentrations were used for every PNPase construct (1:1 and 1:3 RNA:PNPase trimer molar ratio), represented by a concentration bar. (D and E) RNA half-life experiments to determine RyhB and CyaR sRNA stabilities in an E. coli strain expressing a 3X-FLAG tagged construct of PNPase WT and mutants. RyhB and CyaR signal intensities were quantified using northern blots and normalized to their corresponding loading controls (SsrA). sRNA decay curves were generated by fitting the normalized signal intensities for each time point. Points and error bars in the curves represent the means and the standard errors (SEM) of at least three independent experiments. Northern blots for RyhB and CyaR half-life measurements corresponding to RNA stability curves are shown and values tabulated in . (F and G) Cell extracts prepared from late exponential phase cultures of E. coli strains expressing WT PNPase, or FLAG-tagged PNPase WT and mutants were used to assess coprecipitation of sRNAs, which were analyzed using northern blot. (G) Fold enrichment of a given RNA upon immunoprecipitation was determined by first calculating the signal intensity per microgram of RNA for the input and the elution from the northern blots in (F). The normalized elution signal was then divided by the input signal. An untagged wild-type strain (WT) was used as a control for data presented in (F) and (G). S1x2: PNPase K657A, R658A; KHx2: PNPase K566A, K571A; S1x4: PNPase R681A, Q682A, R684A, R686A.

Article Snippet: All strains used in this study are derivatives of E. coli K12 strain MG1655 (RRID:Addgene_61440) or BL21DE3.

Techniques: Electrophoretic Mobility Shift Assay, Construct, Concentration Assay, Expressing, Northern Blot, Generated, Immunoprecipitation, Control

Journal: Molecular Cell

Article Title: A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation

doi: 10.1016/j.molcel.2021.05.032

Figure Lengend Snippet:

Article Snippet: All strains used in this study are derivatives of E. coli K12 strain MG1655 (RRID:Addgene_61440) or BL21DE3.

Techniques: Virus, Recombinant, Software