Kinetic and Thermodynamic Analysis of the Interaction between TRAP (trp RNA-binding Attenuation Protein) of Bacillus subtilis and trp Leader RNA

Chris Baumann,John Otridge,Paul Gollnick

doi:10.1074/jbc.271.21.12269

Chris Baumann, John Otridge + Show 1 more

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https://doi.org/10.1074/jbc.271.21.12269

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Abstract

In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 +/- 1.3 x 10(9) m-1 (Kd = 0.12 +/- 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mM L-tryptophan at 37 degrees C. The temperature dependence of Kapp was somewhat unexpected demonstrating that the delta H of the interaction is highly unfavorable at + 15.9 kcal mol-1. Therefore, the interaction is completely driven by a delta S of +97 cal mol-1 K-1. The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNA-TRAP.tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (> 40 microM) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.

Highlights

In Bacillus subtilis, the genes for tryptophan biosynthesis are regulated in response to tryptophan by an RNA-binding protein called TRAP
Based on the structure of the TRAP endecamer and the multiple trinucleotide repeats in its RNA targets, we have proposed that upon binding, the RNA wraps around the protein such that each G/UAG repeat interacts with one subunit or a combination of two adjacent subunits of the TRAP 11-mer [6]
Most of the RNA (Fig. 2, solid line, closed circles) was in fractions 14 –19 which contained the TRAP1⁄7RNA complex, whereas TRAP was found in two peaks (Fig. 2, solid line, closed squares), one corresponding to the TRAP1⁄7RNA complex and the second corresponding to the uncomplexed TRAP

Summary

Introduction

In Bacillus subtilis, the genes for tryptophan biosynthesis are regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein; Refs. 1–3). In the presence of L-tryptophan, TRAP binds the nascent trp leader transcript [4] at a series of 11 G/UAG repeats between residues ϩ36 and ϩ91 Control of trpE translation is believed to be mediated by TRAP binding to the same series of G/UAG repeats in the leader segment of trp mRNAs that have escaped termination at the attenuator. This binding is thought to alter the RNA secondary structure so as to sequester the trpE ribosome binding site in a stem-loop structure, reducing translation initiation [2]. TRAP contains no sequences with similarity to other characterized RNAbinding motifs [14]

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