Extensive backbone dynamics in the GCAA RNA tetraloop analyzed using 13C NMR spin relaxation and specific isotope labeling.
Conformational dynamics play a key role in the properties and functions of proteins and nucleic acids. Heteronuclear NMR spin relaxation is a uniquely powerful site-specific probe of dynamics in proteins and has found increasing applications to nucleotide base side chains and anomeric sites in RNA. Applications to the nucleic acid ribose backbone, however, have been hampered by strong magnetic coupling among ring carbons in uniformly 13C-labeled samples. In this work, we apply a recently developed, metabolically directed isotope labeling scheme that places 13C with high efficiency and specificity at the nucleotide ribose C2' and C4' sites. We take advantage of this scheme to explore backbone dynamics in the well-studied GCAA RNA tetraloop. Using a combination of CPMG (Carr-Purcell-Meiboom-Gill) and R(1rho) relaxation dispersion spectroscopy to explore exchange processes on the microsecond to millisecond time scale, we find an extensive pattern of dynamic transitions connecting a set of relatively well-defined conformations. In many cases, the observed transitions appear to be linked to C3'-endo/C2'-endo sugar pucker transitions of the corresponding nucleotides, and may also be correlated across multiple nucleotides within the tetraloop. These results demonstrate the power of NMR spin relaxation based on alternate-site isotope labeling to open a new window into the dynamic properties of ribose backbone groups in RNA.
Johnson
James E
JE
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Hoogstraten
Charles G
CG
D001483:Base Sequence; D018360:Crystallography, X-Ray; D055029:Inverted Repeat Sequences; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics*; D012265:Ribonucleotides / Q000737:chemistry; D013997:Time Factors
D012265:Ribonucleotides; D012313:RNA