Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of Hepatitis A virus

Two RNA fragments from the region just upstream of the internal ribosome en try site of Hepatitis A virus (HAV) were studied a 35mer (HAV-35), U-5(4) C -3 U-3 C-3 U-4 C-3 U-3 C-2 UAU(2) C-3 U-3(3'), and a 23mer (HAV-23), U-5'(4 ) C-3 U-3 C-3 U-4 C-3 U-3(3'). Secondary structural predictions and nucleas e digestion patterns obtained with genomic RNAs suggested that they link tw o stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conv entional WC secondary structure, but do fold to form a condensed, stacked ' domain', To obtain more information, folding of HAV-23 and -35 RNA fragment s was characterized using H-1 nuclear magnetic resonance, in H2O as a funct ion of pH and temperature, circular dichroism as a function of NaCl concent ration, pH and temperature, and square-wave voltammetry as a function of pH , The results indicate that these oligonucleotides form intramolecular stru ctures that contain transient U.U base pairs at pH 7 and moderate ionic str ength (100 mM NaCl), This folded structure becomes destabilized and loses t he U.U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with so lvent protons (exchange lifetimes shorter than 1 ms), so the structure cont ains few if any C.C-H(+) base pairs at neutral pH, but can apparently form them at pH values below 6, We present a series of possible models in which chain folding draws the strand termini closer together, possibly sewing to pull the attached WC hairpin domains together and providing a functional ad vantage by nucleating reversible formation of a more viable RNA substrate.