Modulating TRAP-mediated transcription termination by AT during transcription of the leader region of the Bacillus subtilis trp operon

Shraddha Sharma,Paul Gollnick

doi:10.1093/nar/gku211

Abstract

An 11-subunit protein called trp RNA binding Attenuation Protein (TRAP) controls attenuation of the tryptophan biosynthetic (trpEDCFBA) operon in Bacillus subtilis. Tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the 5′ leader region of trp mRNAs, and downregulates expression of the operon by promoting transcription termination prior to the structural genes. Anti-TRAP (AT) is an antagonist that binds to tryptophan-activated TRAP and prevents TRAP from binding to RNA, thereby upregulating expression of the trp genes. AT forms trimers, and multiple trimers bind to a TRAP 11mer. It is not known how many trimers must bind to TRAP in order to interfere with RNA binding. Studies of isolated TRAP and AT showed that AT can prevent TRAP from binding to the trp leader RNA but cannot dissociate a pre-formed TRAP-RNA complex. Here, we show that AT can prevent TRAP-mediated termination of transcription by inducing dissociation of TRAP from the nascent RNA when it has bound to fewer than all 11 (G/U)AG repeats. The 5′-most region of the TRAP binding site in the nascent transcript is most susceptible to dissociation from TRAP. We also show that one AT trimer bound to TRAP 11mer reduces the affinity of TRAP for RNA and eliminates TRAP-mediated transcription termination in vitro.

Highlights

Regulating gene expression by transcription attenuation is a common strategy employed by bacteria [1]
Binding studies using purified AT, trp RNA binding Attenuation Protein (TRAP) and trp leader RNA have shown that AT functions by directly competing with RNA for binding to TRAP [17,19]. These studies indicate that AT cannot induce RNA to dissociate after it has bound to TRAP [17]. These TRAP-RNA complexes may not accurately reflect the situation in vivo where TRAP must bind to the nascent trp transcript during transcription of the leader region in order to regulate transcription of the trp operon [2]
The goal of the studies presented here was to determine whether AT can modulate TRAP-mediated control of attenuation after TRAP has initiated binding to the 11 triplet repeats of the binding site on nascent RNA

Summary

Introduction

Regulating gene expression by transcription attenuation is a common strategy employed by bacteria [1]. In Bacillus subtilis the trp RNA binding Attenuation Protein (TRAP) regulates transcription of the trpEDCFBA operon through an attenuation mechanism in response to changes in the intracellular concentration of tryptophan [2]. When intracellular tryptophan levels are high, TRAP is activated to bind to 11(G/U)AG triplet repeats in the 5 leader region of the trp mRNA. Each (G/U)AG repeat interacts with Glu and Lys 37 from one subunit, and Lys 56 and Arg 58 from the adjacent subunit of TRAP [4,5]. The presence of TRAP bound to the nascent trp transcript induces transcription termination prior to the structural genes of the operon [6,7,8,9]

Methods

Results

Conclusion