Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA.
Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli. Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E. coli as well as eukaryotes. To elucidate its mechanism, E. coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer. The 23 S rRNAs, with an A260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 nM at 37 degrees C and a subsequent treatment with aniline. However, they were not affected by ricin A-chain under the same conditions. The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing. Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E. coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing. As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E. coli 23 S RNA resulting in the inactivation of the ribosome.
Habuka
N
N
Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa.
Miyano
M
M
Kataoka
J
J
Noma
M
M
D001483:Base Sequence; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D009690:Nucleic Acid Conformation; D010940:Plant Proteins / Q000378:metabolism / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S / Q000187:drug effects* / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism
D004269:DNA, Bacterial; D006027:Glycosides; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C059351:MAP protein, Mirabilis jalapa