A quadruple mutant T4 RNA hairpin with the same structure as the wild-type translational repressor

The solution structure of a 16-nucleotide RNA hairpin, 5'-GCCUAG[CAAC]CUGGGC (loop bases in square brackets), has been determined by proton, phosphorus, and carbon (natural abundance) nuclear magnetic resonance (NMR) spectroscopy. This RNA tetraloop hairpin varies in four loop nucleotides from the wild-type T4 RNA hairpin (with eight loop nucleotides) involved in the translational repression of bacteriophage T4 DNA polymerase. Despite the differences in their sequence and proposed secondary structures, these two hairpins bind T4 DNA polymerase with equal affinity. The NMR spectra of the mutant hairpin indicate that its stem is extended in comparison to that of the wild-type hairpin by the formation of two additional Watson-Crick base pairs. The NMR data provide a precisely defined structure for the mutant hairpin with an average root mean square deviation of approximately 0.7 A for all 16 residues in the molecule. The structure of the mutant loop is very similar to that determined previously for the wild-type hairpin. The three loop bases that are conserved between the mutant and wild-type hairpins point out in solution with the groups capable of hydrogen bond formation exposed to the solution. This is exactly what was seen for the wild-type hairpin. Also, unusual, long-range NOEs, loop hydrogen bonds, and even the position at which the loop bends are common features between the two loops. This explains how two different hairpins, by adopting similar three-dimensional structures, have the same affinity for the DNA polymerase.