RNA Structure Inhibits the TRAP (trpRNA-binding AttenuationProtein)-RNA Interaction

Sandhya Xirasagar,Matthew B Elliott,Wilmin Bartolini,Paul Gollnick,Philip A Gottlieb

doi:10.1074/jbc.273.42.27146

Sandhya Xirasagar, Matthew B Elliott + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.42.27146

Copy DOI

Abstract

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

Highlights

IntroductionThe Bacillus subtilis TRAP1 protein (trp RNA-binding attenuation protein) negatively regulates expression of the tryptophan biosynthetic (trp) genes in response to tryptophan [1, 2]
The Bacillus subtilis TRAP1 protein negatively regulates expression of the tryptophan biosynthetic genes in response to tryptophan [1, 2]
As well as mutagenesis and footprinting experiments have suggested that the spacer nucleotides do not directly interact with the protein [8, 9]

Summary

Introduction

The Bacillus subtilis TRAP1 protein (trp RNA-binding attenuation protein) negatively regulates expression of the tryptophan biosynthetic (trp) genes in response to tryptophan [1, 2]. TRAP associates with two specific RNA targets and regulates transcription of the trpEDCFBA operon [1,2,3,4] as well as translation of trpE [5] and trpG [6, 7]. The RNA-binding sites for TRAP contain multiple GAG or UAG (rarely AAG) triplet repeats, generally separated by two or three spacer nucleotides [8, 9]. The trp leader RNA contains 11 triplet repeats and the binding site in trpG contains 9 repeats. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

Objectives

Results

Conclusion