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Volume 272, Number 32, Issue of August 8, 1997 pp. 19863-19869
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis Binds to Unstacked trp Leader RNA

(Received for publication, January 24, 1997, and in revised form, April 16, 1997)

From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260

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 °C and 47 °C and then decreasing from 47 °C to 67 °C. Differential scanning calorimetry and circular dichroism spectroscopy demonstrate that TRAP is highly thermostable with few detectable changes in the structure between 25 °C and 70 °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.

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