PROBING REGA RNA INTERACTIONS USING ELECTROSPRAY-IONIZATION FOURIER-TRANSFORM ION-CYCLOTRON RESONANCE-MASS SPECTROMETRY/

The interactions of bacteriophage T4 regA protein, a unique translatio nal regulator, with RNAs of various size and sequence were studied usi ng electrospray ionization-Fourier transform ion cyclotron resonance-m ass spectrometry. Using very gentle interface conditions, regA/RNA com plexes with a 1:1 binding stoichiometry were observed for all four tar get RNAs studied, consistent with solution binding studies. Competitiv e binding of target RNAs and their degradation products with regA demo nstrated that the loss of a single nucleotide resulted in a dramatic c hange in binding affinity in some cases. Competitive binding of regA w ith four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments. The use of sustained off-resonance irradiation for collisionally induced diss ociation of a regA/RNA complex suggested the potential for directly ob taining information regarding the regA binding domain. (C) 1998 Academ ic Press.