title: Nucleotide contributions to the structural integrity and DNA replication initiation activity of noncoding y RNA.
abstract: Noncoding Y RNAs are small stem-loop RNAs that are involved in different cellular processes, including the regulation of DNA replication. An evolutionarily conserved small domain in the upper stem of vertebrate Y RNAs has an essential function for the initiation of chromosomal DNA replication. Here we provide a structure-function analysis of this essential RNA domain under physiological conditions. Solution state nuclear magnetic resonance and far-ultraviolet circular dichroism spectroscopy show that the upper stem domain of human Y1 RNA adopts a locally destabilized A-form helical structure involving eight Watson-Crick base pairs. Within this helix, two G:C base pairs are highly stable even at elevated temperatures and therefore may serve as clamps to maintain the local structure of the helix. These two stable G:C base pairs frame three unstable base pairs, which are located centrally between them. Systematic substitution mutagenesis results in a disruption of the ordered A-form helical structure and in the loss of DNA replication initiation activity, establishing a positive correlation between folding stability and function. Our data thus provide a structural basis for the evolutionary conservation of key nucleotides in this RNA domain that are essential for the functionality of noncoding Y RNAs during the initiation of DNA replication.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica , 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Iren
authors.initials: I
authors.lastname: Wang
authors.forename: Madzia P
authors.initials: MP
authors.lastname: Kowalski
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Langley
authors.forename: Raphaël
authors.initials: R
authors.lastname: Rodriguez
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
authors.forename: Shang-Te Danny
authors.initials: ST
authors.lastname: Hsu
authors.forename: Torsten
authors.initials: T
authors.lastname: Krude
mesh_terms: D020029:Base Pairing; D002942:Circular Dichroism; D004261:DNA Replication; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D022661:RNA, Untranslated / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D009711:Nucleotides; D022661:RNA, Untranslated