Role of Bacillus subtilis RNase J1 Endonuclease and 5′-Exonuclease Activities in trp Leader RNA Turnover

Gintaras Deikus,Ciarán Condon,David H Bechhofer

doi:10.1074/jbc.m801461200

Abstract

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.

Highlights

We had shown previously that endonuclease cleavage around nt 100 of trp leader RNA was responsible for initiation of decay [16]
To study whether RNase J1 was involved in trp leader RNA turnover, strains were constructed that carried the high copy number plasmid pGD5 [16] containing the trp leader region and a copy of the mtrB gene and that conditionally expressed RNase J1 under control of the IPTG-inducible pspac promoter or had a deletion of the gene encoding RNase J2
If the in vitro results obtained with TRAP-bound RNA reflected events occurring in vivo, we predicted that, in a strain grown in the presence of tryptophan, RNase J1 would cleave in the single-stranded regions of trp leader RNA on either side of the TRAP binding site, generating an RNA fragment about the size of this region (56 nt)

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains—The “wild-type” B. subtilis strain for in vivo experiments was BG581, which is trpC2 thr-5 and carries plasmid pGD5, a high copy number plasmid that contains the trp leader region and the mtrB gene [16]. For 5Ј-triphosphate end labeling of trp leader RNA, transcription was done in the presence of [␥-32P]GTP. RNA oligonucleotides, bearing a 5Ј-hydroxyl end, were obtained from Integrated DNA Technologies and were labeled at the 5Ј-end (monophosphate) with polynucleotide kinase and at the 3Ј-end by T4 RNA ligase, as above for full-length trp leader RNA, and were purified from 9% denaturing polyacrylamide gels. For the RNA oligonucleotide with the labeled triphosphate end, RNA was synthesized by T7 RNA polymerase transcription in the presence of [␥-32P]GTP and purified from a 9% polyacrylamide gel. High resolution Northern blot analysis was performed using a 7% denaturing polyacrylamide gel and electroblotting at 20 volts overnight, followed by an additional 60 min at 30 volts.

RESULTS

We considered the possibility that

DISCUSSION