In Vitro Transcription Translation Reaction, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA"
Article Title: Photorhabdus antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of 23S ribosomal RNA
Journal: Nucleic Acids Research
Figure Legend Snippet: Tre23 inhibits protein synthesis. ( A ) Phase contrast and fluorescence microscopy recordings of E. coli cells constitutively producing short-lived green fluorescent protein (GFP-LVA) and the Tre23 toxin from the low-copy number pNDM220 vector, before and 100 min post toxin induction. Cells bearing the empty pNDM220 vector (Ø) were used as control. ( B ) Replication, transcription and translation rates before (time 0) and 60 min after the induction of Tre23 toxin from pNDM220 vector (red line) as compared to control carrying empty vector (black line) were measured by the incorporation (in count per minute, cpm) of [ 3 H] thymidine, [ 3 H] uridine and [ 35 S] methionine, respectively. ( C ) Coupled in vitro transcription-translation reaction. In vitro transcription/translation of the Strep-tagged GFP reporter protein was estimated by immunodetection. Reactions were supplemented with NAD + , Tre23 and Tri23, as indicated. In the last reaction, Tri23 was added 1 h after incubating the reaction with Tre23 (aft) and reaction proceeded for 2 h. ( D ) Tre23-Tri23 co-purification. Total cell lysates of E. coli BL21(DE3) cells producing FLAG-tagged Tri23 (Tri23 FL ) alone or co-producing Strep-tagged Tre23 (Tre23 ST ) (T) were subjected to purification on streptactin agarose beads followed by immunoprecipitation on anti-FLAG affinity gel (IP). Proteins were separated by SDS-PAGE and stained by Coomassie (upper panel) or immunodetected using anti-Strep and anti-FLAG antibodies (middle and bottom panels, respectively).
Techniques Used: Fluorescence, Microscopy, Low Copy Number, Plasmid Preparation, In Vitro, Immunodetection, Copurification, Purification, Immunoprecipitation, SDS Page, Staining
Figure Legend Snippet: Tre23 ADP-ribosylates the 23S RNA of actively translating ribosomes. ( A and B ) Native PAGE (A) and SDS-PAGE (B) analyses of in vitro couple translation-transcription reaction supplemented with 6-biotin-17-NAD + ( biot NAD + ) and with Tre23, as indicated. Samples were stained with Coomassie blue (left panels) or detected using streptavidin-AP (right panels). Mass spectrometry analysis of the indicated band identified subunits of 30S and 50S ribosome (Ribos.). ( C ) RNA extracted from of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), Tre23 and Tri23, as indicated were stained with ethidium bromide (EtBr, left panel) or detected using streptavidin-AP conjugate (right panel). The positions of tRNAs, 16S and 23S ribosomal RNAs are indicated. ( D ) Western-blot analyses with anti-Strep antibodies of GFP-strepII in vitro transcription-translation in presence of ribosomes purified from wild-type E. coli cells carrying empty pNDM220 vector (Ø), or cells producing Tre23. Ribosomes (R) included in the coupled transcription-translation kit were used as control. Ribosome profile changes over time after toxin induction are shown in Supplementary Figure S2 . ( E ) RNA analyses of in vitro transcription-translation reactions supplemented with 6-biotin-17-NAD + ( biot NAD + ), and Tre23, as indicated, using ribosomes purified from wild-type E. coli cells bearing empty vector (Ø), or cells producing Tre23. RNAs were stained with EtBr (left panel) or detected using streptavidin-AP (right panel). ( F ) EtBr staining (left panel) and Streptavidin-AP (right panel) RNA analyses of purified ribosomal RNAs (rRNA), or purified ribosomes alone or supplemented with translation factors (F) or translation reagents (tRNAs, amino acids, nucleotides; A), in presence of 6-biotin-17-NAD + ( biot NAD + ) and Tre23, as indicated.
Techniques Used: Clear Native PAGE, SDS Page, In Vitro, Staining, Mass Spectrometry, Western Blot, Purification, Plasmid Preparation