RNase-stable RNA: conformational parameters of the nucleic acid backbone for binding to RNase T1.
An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.
Greiner-Stöffele
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Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Germany.
Förster
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HH
Hofmann
H J
HJ
Hahn
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U
D001483:Base Sequence; D003198:Computer Simulation; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C098798:SYBR Green II; D012313:RNA; D006163:Ribonuclease T1