PROTON NMR AND STRUCTURAL FEATURES OF A 24-NUCLEOTIDE RNA HAIRPIN

The three-dimensional conformation of a 24-nucleotide variant of the R NA. binding sequence for the coat protein of bacteriophage R17 has bee n analyzed using NMR, molecular dynamics, and energy minimization, The imino proton spectrum is consistent with base pairing requirements fo r coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2 D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including d econvolution and path analysis methods. An average of 21 distance cons traints per residue were used in molecular dynamics calculations to ob tain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to C15 i n the upper stem and loop appears to be flexible. Several of these res idues have a large fraction of S-puckered ribose rings, rather than th e N-forms characteristic of RNA duplexes. There is considerable variat ion in the low-energy loop conformations that satisfy the distance con straints at this preliminary level of refinement. The Shine-Dalgarno r ibosome binding site is exposed, and only two apparently weak base pai rs would have to break for the 16S ribosomal RNA to bind and the ribos ome to initiate translation of the replicase gene. Although the loop f orm must be regarded as tentative, the known interaction sites with th e coat protein are easily accessible from the major groove side of the loop.