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 entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the 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 solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+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 serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.
Hardin
C C
CC
Department of Biochemistry and Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA. hardin@bchserver.bch.ncsu.edu
Sneeden
J L
JL
Lemon
S M
SM
Brown
B A
BA
Guenther
R H
RH
Sierzputowska-Gracz
H
H
D020029:Base Pairing; D001483:Base Sequence; D002942:Circular Dichroism; D006507:Hepatovirus / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011199:Potentiometry; D014176:Protein Biosynthesis; D011742:Pyrimidine Nucleotides / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism
D011742:Pyrimidine Nucleotides; D012367:RNA, Viral