Bacillus subtilis trp Leader RNA: RNase J1 ENDONUCLEASE CLEAVAGE SPECIFICITY AND PNPase PROCESSING

Gintaras Deikus,David H Bechhofer

doi:10.1074/jbc.m109.015875

Abstract

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3' transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3'-to-5' exonuclease processing from the native 3' end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5' tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5' end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.

Highlights

Protective features of the 5Ј and 3Ј ends of prokaryotic mRNAs explain why these RNA molecules are not degraded immediately by the multiple ribonucleases that are present in a prokaryotic cell
A general model for the initiation of mRNA decay in prokaryotes, which is based on numerous studies in Escherichia coli, is as follows: initiation of decay occurs by an endonucleolytic cleavage in the body of the message
It was believed initially that RNase J1 was exclusively an endonuclease, more recently it was discovered that RNase J1 has, in addition, a 5Ј-to-3Ј exonuclease activity [2, 3], which requires a 5Ј-monophosphate end. This discovery is the basis for an alternative to the E. coli model for mRNA decay that would apply to organisms that express RNase J1: mRNA could be degraded directly from the 5Ј end, after pyrophosphate removal, or RNase J1 could act as both an endonuclease to cleave the initial transcript and as a 5Ј exonuclease on the downstream product of such cleavage [2, 20]

Summary

Introduction

Protective features of the 5Ј and 3Ј ends of prokaryotic mRNAs explain why these RNA molecules are not degraded immediately by the multiple ribonucleases that are present in a prokaryotic cell. Wild-type sized trp leader RNA was detected for each of the constructs, indicating that the mutant sequences did not affect conformation of the transcription terminator structure or binding of TRAP, which is required for termination.

Results

Conclusion