The Bacillus subtilis TRAP Protein Can Induce Transcription Termination in the Leader Region of the Tryptophan Biosynthetic (trp) Operon Independent of the trp Attenuator RNA

Natalie M Mcadams,Paul Gollnick,Dirk-Jan Scheffers

doi:10.1371/journal.pone.0088097

Natalie M Mcadams, Paul Gollnick + Show 1 more

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https://doi.org/10.1371/journal.pone.0088097

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Journal: PloS one	Publication Date: Feb 4, 2014
Citations: 10	License type: CC BY 4.0

Affiliation: University at Buffalo, State University of New York

Abstract

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. When intracellular tryptophan levels are high, the TRAP protein binds to the 5′ leader region of the nascent trp mRNA and induces transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator) can form in the leader region RNA. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. However, recent studies have shown that the attenuator is not an effective intrinsic terminator. From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Hence we have further examined the role of the attenuator RNA in TRAP-mediated transcription termination. TRAP was found to cause efficient transcription termination in the trp leader region in vivo when the attenuator was mutated or deleted. However, TRAP failed to induce transcription termination at these mutant attenuators in a minimal in vitro transcription system with B. subtilis RNA polymerase. Further studies using this system showed that NusA as well as the timing of TRAP binding to RNA play a role in the observed differences in vivo and in vitro.

Highlights

Expression of the trpEDCFBA operon, which contains six of the seven genes required for tryptophan biosynthesis in B. subtilis, is regulated by the trp RNA-binding attenuation protein (TRAP) [1,2]
While it contains features in common with intrinsic terminators, including a stem-loop structure followed by a U-stretch (Figure 2A), efficient transcription termination at the trp attenuator only occurs when TRAP is bound to the nascent trp transcript [12]
Since the goal of our RNase protection assays was to determine whether TRAP induces termination at the same location in Wild Type (WT) and several mutant trp leader regions, we only examined trp transcripts produced from cells grown in excess tryptophan

Summary

Introduction

Expression of the trpEDCFBA (trp) operon, which contains six of the seven genes required for tryptophan biosynthesis in B. subtilis, is regulated by the trp RNA-binding attenuation protein (TRAP) [1,2]. Transcription of this operon is regulated by an attenuation mechanism. Two mutually exclusive RNA secondary structures called the antiterminator and the terminator (attenuator), which form in the 203 nt 59 leader region of the trp transcript upstream of trpE, are proposed to control transcription of the operon [3,4]. When tryptophan is limiting, TRAP does not bind RNA, and the antiterminator structure forms, which allows transcription of the trp genes (Figure 1) [5]

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