Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA.
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.
Baumann
C
C
Department of Biological Sciences, State University of New York at Buffalo 14260, USA.
Otridge
J
J
Gollnick
P
P
D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012492:Salts; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; C079092:MtrB protein, Bacteria; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012492:Salts; D014157:Transcription Factors; D014364:Tryptophan