Abstract
TRAP (trp RNA-binding attenuation protein) is a tryptophan-activated RNA-binding protein that regulates expression of the trp biosynthetic genes by binding to a series of GAG and UAG trinucleotide repeats generally separated by two or three spacer nucleotides. Previously, we showed that TRAP contains 11 identical subunits arranged in a symmetrical ring. Based on this structure, we proposed a model for the TRAP.RNA interaction where the RNA wraps around the protein with each repeat of the RNA contacting one or a combination of two adjacent subunits of the TRAP oligomer. Here, we have shown that RNAs selected in vitro based on their ability to bind tryptophan-activated TRAP contain multiple G/UAG repeats and show a strong bias for pyrimidines as the spacer nucleotides between these repeats. The affinity of the TRAP.RNA interaction displays a nonlinear temperature dependence, increasing between 5 degrees C and 47 degrees C and then decreasing from 47 degrees C to 67 degrees C. Differential scanning calorimetry and circular dichroism spectroscopy demonstrate that TRAP is highly thermostable with few detectable changes in the structure between 25 degrees C and 70 degrees C, suggesting that the temperature dependence of this interaction reflects changes in the RNA. Results from circular dichroism and UV absorbance spectroscopy support this hypothesis, demonstrating that trp leader RNA becomes unstacked upon binding TRAP. We propose that the bias toward pyrimidines in the spacer nucleotides of the in vitro selected RNAs represents the inability of Us and Cs to form stable base stacking interactions, which allows the flexibility needed for the RNA to wrap around the TRAP oligomer.
Highlights
The tryptophan biosynthetic genes of Bacillus subtilis are negatively regulated in response to the intracellular level of L-tryptophan by a tryptophan-activated RNA-binding protein called TRAP1
Effects of Temperatures on the TRAP1⁄7trp Leader RNA Interaction—In light of the indication that base stacking interactions play a role in the TRAP1⁄7RNA interaction, we examined the affinity of TRAP for RNA 36 –92 at temperatures up to 67 °C (Fig. 4)
The RNAs that we selected in vitro based on their ability to bind TRAP all contain multiple GAG and/or UAG repeats confirming the importance of these trinucleotides for TRAP binding
Summary
Plasmids and Strains—Plasmids used in this study were propagated in Escherichia coli JM107. Plasmid pCB1, which contains 11 repeats of the sequence 5Ј-GAGAA-3Ј under control of a T7 promoter, was designed based on work described previously [13] and generated as follows. RNA Synthesis and TRAP Purification—The in vitro synthesis of 32P-labeled trp leader RNA using T7 RNA polymerase has been described previously [9]. In Vitro Selection of RNAs That Bind TRAP—SELEX was performed as outlined by Tuerk [19] using an oligonucleotide pool containing 25 random positions (25N; Ref. 20). We found that the observed dissociation constant (Kobs) for TRAP and an RNA containing the first five G/UAG repeats of the trp leader transcript (RNA 2– 64) is 100 nM (data not shown). RNAs that bound to TRAP were selected by filter binding as described by Tuerk [19]. All data were normalized to the absorbance at 15 °C
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
