Initiation of decay of Bacillus subtilis trp leader RNA

Gintaras Deikus,David H Bechhofer

doi:10.1074/jbc.m702747200

Abstract

Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3' end of trp leader RNA and PNPase digestion of trp leader RNA from the 3' end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.

Highlights

Ing of the TRAP complex allows a stem-loop transcription terminator structure to form, which consists of nt 108 –133 of the trp leader sequence
For the trp system to be regulated by TRAP, sufficient free TRAP complex needs to be present to bind to newly made trp leader RNA, which is constitutively transcribed from the trp promoter
The small size of trp leader RNA makes it an ideal model to study RNA turnover. (In this report, “trp leader RNA” refers to the 139-nt RNA that is the result of transcription termination in the trp leader sequence.) trp leader RNA is rapidly degraded in a wild-type strain with a half-life of about 1 min, significantly shorter than the average half-life of B. subtilis mRNAs [7]

Summary

Initiation of decay of Bacillus subtilis trp leader RNA*

The presence of stem-loop structures at the 5Ј and 3Ј ends of trp leader RNA (Fig. 1B), as well as the binding of a large portion of the internal sequence by the TRAP complex, makes it suitable as an object of study, as one would predict that there are few available internal sites in this molecule at which decay could initiate. Because it was known from our previous study that PNPase is responsible for trp leader RNA degradation, we studied PNPase activity on trp leader RNA in vitro. These experiments led to a hypothesis concerning initiation of trp leader RNA turnover in vivo, which was confirmed by analysis of decay of mutant trp leader RNA

EXPERIMENTAL PROCEDURES

Findings

DISCUSSION