Role of Bacillus subtilis RNase J1 endonuclease and 5'-exonuclease activities in trp leader RNA turnover.
The 140-nucleotide trp leader RNA, which is formed by transcription termination under conditions of high intracellular tryptophan, was used to study RNA turnover in Bacillus subtilis. We showed in vivo that the amount of endonuclease cleavage at approximately nucleotide 100 is decreased under conditions where RNase J1 concentration is reduced. In addition, under these conditions the level of 3'-terminal RNA fragments, which contain the strong transcription terminator structure, increases dramatically. These results implicated RNase J1 in the initiation of trp leader RNA decay as well as in the subsequent steps leading to complete turnover of the terminator fragment. To confirm a direct role for RNase J1, experiments were performed in vitro with various forms of trp leader RNA and 3'-terminal RNA fragments. Specific endonuclease cleavages, which were restricted to single-stranded regions not bound by protein, were observed. Degradation of the 3'-terminal fragment by the 5' to 3'-exonuclease activity of RNase J1 was also demonstrated, although the presence of strong secondary structure impeded RNase J1 processivity to some extent. These results are consistent with a model for mRNA decay in Bacillus subtilis whereby the downstream products of RNase J1 endonucleolytic cleavage become substrates for the 5' to 3'-exoribonuclease activity of the enzyme.
Deikus
Gintaras
G
Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA.
Condon
Ciarán
C
Bechhofer
David H
DH
D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D015152:Blotting, Northern; D002384:Catalysis; D004722:Endoribonucleases / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D009154:Mutation; D043264:Phosphodiesterase I / Q000378:metabolism*; D012313:RNA / Q000737:chemistry*; D012260:Ribonucleases / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity; D013997:Time Factors
D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases; D043264:Phosphodiesterase I