| The production of Antirepressor protein (Ant) during lytic development in the lambdoid bacteriophage P22 is controlled in part by an antisense RNA, sar RNA. The effectiveness of antisense control is related to the pairing rate of sar RNA to ant mRNA. Thus, I have examined the pairing of sar RNA to ant mRNA in detail. Three aspects of the pairing reaction were considered: RNA structure, reaction pathway, and ionic dependence.;The structures of sar RNA and ant RNA were examined by limited RNase digestion over a range of Mg$sp{2+}$ concentrations. Sar RNA forms two hairpins that are present at all Mg$sp{2+}$ concentrations (Mg$sp{2+}$-independent hairpins). Ant RNA forms four hairpins and a multi-branched loop structure. One of the hairpins is Mg$sp{2+}$-dependent; the rest are Mg$sp{2+}$-independent. In addition, there is evidence for Mg$sp{2+}$-dependent tertiary structure in both RNAs. The precise nature of the tertiary interactions in sar RNA and ant RNA has not been determined.;The pairing pathway was investigated by measuring kinetic parameters for three sequential reaction steps, using electrophoretic mobility shift (gel-shift) assays and RNase protection assays. The three reaction steps are described below. First, reversible base pairs between nucleotides contained in both sar RNA loops and their complements in ant RNA form. Second, stable complex formation initiates between unpaired nucleotides in sar RNA and ant RNA. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin and duplex propagation then occur, resulting in a partially paired intermediate. Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin.;The ionic dependence of the pairing reaction was investigated by measuring kinetic parameters in the presence of various ions, using a gel-shift assay. Maximal second-order rate constants require binding of at least two Mg$sp{2+}$ ions. Both binding sites exhibit ionic specificity. |
