Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: srn_3610_sprC expression increases in the absence of SarA. ( A ) Srn_3610_sprC expression in different Staphylococcus aureus strains lacking transcription factors (TFs). Northern blot analysis of the Srn_3610_sprC and transfer-messenger RNA (tmRNA) transcripts in the S. aureus HG003 strain and in isogenic mutants for the SarA protein family. All samples were harvested after 5 h of growth. A total of 10 μg of total RNA were analyzed, and the Digoxigenin method (Roche) was used to reveal Srn_3610_sprC and tmRNA (loading control). ( B ) Northern blot analysis of Srn_3610_SprC and tmRNA transcript expression in S. aureus HG003 and isogenic HG003Δ sarA . Samples were harvested at the specified time point, and tmRNA was used as a control. In a graph showing optical density as a function of time, the growths of HG003 (diamonds) and HG003Δ sarA (triangles) are represented in a semi-logarithmic scale.

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: Expressing, Northern Blot

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA represses srn_3610_sprC expression. ( A ) Staphylococcus aureus HG003 and HG003Δ sarA strains were co-transformed with: pCN36/pCN41c empty vectors (lanes 1 and 5); pCN36/pCN41c - P sprC (lanes 2 and 6); pCN36-SarA/pCN41c (lanes 3 and 7); and pCN36-SarA/pCN41- P sprC (lanes 4 and 8). Srn_3610_SprC and SarA transcripts levels were assessed by northern blot. A 16S rRNA probe was used to reflect total RNA loading for each lane. In parallel, SarA protein levels were checked by western blot (middle panel) and SYPRO Ruby staining of total protein extracts was done to compare loaded protein levels (bottom). ( B ) Effects of SarA on transcriptional activity of the srn_3610_sprC promoter (P sprC ). S. aureus HG003 wild-type strain (HG003) and a strain lacking sarA (HG003Δ sarA ) were co-transformed with pCN41c or pCN41c-P sprC and either pCN36 or pCN36-SarA. P sprC activity was estimated by measuring ß-lactamase substrate hydrolysis. For each lane, indicated ß-lactamase activity was normalized by subtracting the background signal from the same strain where pCN41c-P sprC was replaced by pCN41c empty vector (not shown). Three independent experiments were done, error bars show ± standard deviation. (Mann–Whitney test; * P < 0.05; *** P < 0.001.).

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: Expressing, Transformation Assay, Northern Blot, Western Blot, Staining, Activity Assay, Plasmid Preparation, Standard Deviation, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: srn _ 9340 possesses a putative binding site for SarA. Srn_3610_sprC belongs to the pathogenicity island SaPIn3. SprC 47 , which can interact with SarA, possesses a 22-nt palindromic sequence (underlined). Blast analysis using sprC 47 as the query led to the discovery of another putative SarA binding site within SaPIn3. This sequence was identified as part of the promoter of another small RNA, Srn_9340.

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA binds srn_9340 on its promoter and represses transcription. ( A ) SarA represses srn_9340 expression. After 5 h of growth, 10 μg of total RNA were obtained from Staphylococcus aureus HG003 and HG003Δ sarA . Srn_9340 expression levels were monitored by northern blot. ( B ) Schematic representation of both the 5′ and 3′ ends of the Srn_9340 transcripts (long and short small RNAs), as determined by rapid amplification of cDNA ends (RACE). ( C ) SarA binds the srn_9340 promoter in vitro . An EMSA was done using 10 fmol of a 32 P-labeled srn_9340 promoter fragment ( srn_9340 P225 ) as a probe in the presence of increasing amounts (0.01–1 pmol) of 6His-tagged SarA. Srn_9340 P225 forms an initial complex with 0.15 pmol of SarA, and a second one at 0.75 pmol. ( D ) SarA specifically binds srn_9340 P225 in vitro . EMSA was done with 10 fmol of 32 P-labeled srn_9340 P225 , 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 P225 ) or non-specific competitors (unlabeled 16S 225 ). ( E ) DNase I footprinting assays were performed in the presence of 10 fmol srn_9340 P225 , 7.5.10 −2 U DNase I and increasing amounts (0.1–0.5 pmol) of 6His-SarA. The region protected by SarA is indicated with a vertical dotted arrow, and the numbers indicate relative positions to the previously determined transcription start site (TSS). Lanes G, A, T and C correspond to sequencing. The nucleotides from −51 to −1 ( srn_9340 51 ) are protected by SarA against DNase I degradation. ( F ) Schematic representation of the DNA probes used for EMSA studies. ( G ) Deletion of the protected SarA sequence from the srn_9340 promoter region abolishes SarA's capacity to bind the srn_9340 promoter. ( H ) A 51 bp protected sequence is sufficient for SarA binding. EMSA were done using 10 fmol srn_9340 51 in the presence of increasing amounts of 6His-tagged SarA (0.25–2 pmol). ( I ) Srn_9340 51 competes with srn_9340 225 for SarA binding. EMSA was performed with 10 fmol of 32 P-labeled srn_9340 P225 , 0.4 or 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340 51 ) or non-specific competitors (unlabeled Random 51 ). ( J ) The 23 bp SarA binding site on srn_9340 225 was confirmed by EMSA done in the presence of 0.5 pmol sarA and with increasing amounts of unlabeled srn_9340 225 or unlabeled srn_9340 225 Δ23 .

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: Expressing, Northern Blot, Rapid Amplification of cDNA Ends, In Vitro, Labeling, Footprinting, Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: In vivo analysis of the srn_3610_sprC and srn_9340 promoters: SarA versus σ A binding. The Staphylococcus aureus HG003Δ sarA strain was transformed either with an empty pCN38 vector (dark gray) or with pCN38-SarA6His (light gray). Chromatin immunoprecipitation (ChIP) experiments were conducted using antibodies for 6His-tag ( A ) or for the σ 70 /σ A RNA polymerase subunit ( B ). DNA enrichment was assessed by qPCR analysis using specific primers for the promoters of srn_3610_sprC (left panel), srn_9340 (middle) or hla (right). Values beneath the bars indicate fold change between specific and non-specific interactions. (Mann–Whitney U test; three independent experiments; * P < 0.05). (A) srn_3610_sprC, srn_9340 and hla promoters co-immunoprecipitate with SarA6His. (B) SarA6His disturbs σ A binding on the srn_3610_sprC promoter.

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: In Vivo, Binding Assay, Transformation Assay, Plasmid Preparation, Chromatin Immunoprecipitation, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus

doi: 10.1093/nar/gkw777

Figure Lengend Snippet: SarA and bacterial RNA polymerase holoenzyme cannot simultaneously bind sprC P267 and srn_9340 P225 . EMSA was done using 10 fmol 32 P-labeled sprC P267 or srn_9340 P225 probes with increasing amounts (2–100 fmol) of purified RNA polymerase. The binding capacity of Escherichia coli RNA polymerase was confirmed by EMSA experiments using sprC P267 ( A ) or srn_9340 P225 ( B ). To demonstrate their binding exclusivity on sprC P267 ( C ) or srn_9340 P225 ( D ), 0.5 pmol purified SarA and 50–100 fmol E. coli RNA polymerase were simultaneously added to EMSA experiments. In vitro transcription was realized using 10 fmol of sprC 456 ( E ) or srn_9340 337 ( F ) and 100 fmol of E. coli RNA polymerase. A total of 5 pmol of the purified TF SarA were added, when indicated.

Article Snippet: In vitro transcription was realized adding 100 fmol of E. coli RNA polymerase (New England Biolabs) to 10 fmol of DNA template ( sprC 456 or srn_9340 337 ), in 5× E. coli RNA polymerase reaction buffer (New England Biolabs) supplementing with nucleoside triphosphate ATP, CTP, GTP (0.75 mM each) and 0.5 μCi of [α- 32 P] UTP.

Techniques: Labeling, Purification, Binding Assay, In Vitro