Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction.
The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically. The pairing reaction proceeds by a three-step pathway. First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM). Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1). Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1). Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters. The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation. Maximal pairing rates are achieved when more than one Mg2+ ion is bound. The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.
Schaefer
K L
KL
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
McClure
W R
WR
D017100:Bacteriophage P22 / Q000235:genetics*; D002412:Cations; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016372:RNA, Antisense / Q000737:chemistry / Q000378:metabolism*
D002412:Cations; D016372:RNA, Antisense; D008274:Magnesium