RNA STRUCTURE INHIBITS THE TRAP (TRP RNA-BINDING ATTENUATION PROTEIN)RNA INTERACTION

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus s ubtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer n ucleotides. Previous mutagenesis experiments have identified three TRA P residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA bind ing. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer, In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)(11)) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of te mperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers co mbined with UV hyperchromicity and circular dichroism. Furthermore, in troducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)(11) RN As stabilized the TRAP-RNA interaction, This effect was associated wit h decreased stability of the RNA structure as measured by circular dic hroism spectroscopy. The precise nature of the structure of the ((G/U) AGCC)(11) RNAs is not known but evidence is presented that it involves noncanonical interactions, We also observed that substitution of Arg- 58 with Lys further reduced the ability of TRAP to interact with struc tured RNAs. Since in vivo function of TRAP may involve binding to stru ctured RNAs, we suggest a potential function for this residue, which i s conserved in TRAP from three different bacilli.