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

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 ؋ 10 9 M ؊1 (K d ‫؍‬ 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°C. The temperature dependence of K app was somewhat unexpected demonstrating that the ⌬H of the interaction is highly unfavorable at ؉15.9 kcal mol ؊1. Therefore, the interaction is completely driven by a ⌬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 RNA⅐TRAP⅐tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (>40 M) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.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). TRAP regulates expression of the trp genes in three ways, all of which involve TRAP binding RNA in a tryptophandependent manner (3). The trp operon (trpEDCFBA), which contains 6 of the 7 genes required for L-tryptophan biosynthesis, is regulated by transcription attenuation within a 204-nucleotide leader region preceding the first structural gene, trpE (1-2). 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 ( Fig. 1; Refs. 5-7). This binding prevents formation of an anti-terminator secondary structure which allows a transcription terminator to form and transcription halts in the leader region (1, 8). When L-tryptophan is limiting, TRAP does not bind, the anti-terminator forms, and the operon is expressed.TRAP also regulates translation of two trp genes, trpE (2, 9) and trpG (10), apparently by two different mechanisms. 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, thus reducing translation initiation (2).trpG is the only tryptophan biosynthetic gene not located within the trp operon and is located within a folic acid biosynthetic operon (11). Regulation of trpG translation occurs wh...

Using Hetero-11-mers Composed of Wild Type and Mutant Subunits to Study Tryptophan Binding to TRAP and Its Role in Activating RNA Binding

“…In addition to affecting the affinity of TRAP for its various RNA targets, the number of tryptophan molecules bound to TRAP may affect the kinetics of the interaction between TRAP and RNA. For example, the dissociation rate of the TRAP-RNA complex is dependent on the concentration of tryptophan (28). This rate is important in vivo because it affects the number of molecules of free TRAP available for binding to its target mRNAs.…”

The mechanism of RNA binding to TRAP: Initiation and cooperative interactions

“…In vitro transcription reactions to prepare 32 P-labeled samples were performed with T7 RNA polymerase and [a-32 P]-ATP, or [a-32 P]-UTP (NEN) as described previously (Baumann et al+, 1996;Xirasagar et al+, 1998;Elliott et al+, 1999) using plasmids linearized with BamHI or HindIII as templates+ All D/RNA chimeras were chemically synthesized as described previously (Elliott et al+, 1999) by the CAMBI Nucleic Acid Facility, State University of New York, Buffalo, New York, and 59-end labeled using [g-32 P]-ATP and polynucleotide kinase (Sambrook et al+, 1989)+…”

The Anti-trp RNA-binding Attenuation Protein (Anti-TRAP), AT, Recognizes the Tryptophan-activated RNA Binding Domain of the TRAP Regulatory Protein

“…TRAP interactions with tryptophan and with RNA have been studied in great detail (13)(14)(15)(16)(17)(18)(19)(20)(21). TRAP is composed of 11 identical subunits arranged as a doughnut-shaped molecule.…”