Isolation and Characterization of a Family of Stable RNA Tetraloops with the Motif YNMG That Participate in Tertiary Interactions

Abstract

RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

“…Thermodynamic parameters are based on the set of Xia et al (44)(45)(46) and Mathews et al (8). Hairpin loop parameters (Tables 1 and 2) are revised on the basis of recent experimental results (47,48) and the previous database of RNA hairpin stabilities (49)(50)(51)(52)(53)(54)(55).…”

Computational approaches for RNA energy parameter estimation

“…Secondly, we collected a thermodynamic set denoted by T-Full that contains data from 1291 optical melting experiments, published in 53 research articles Sugimoto et al 1986Sugimoto et al , 1987Uhlenbeck 1988, 1989;Longfellow et al 1990;SantaLucia et al 1990SantaLucia et al , 1991aAntao et al 1991;He et al 1991;Peritz et al 1991;Antao and Tinoco 1992;Serra et al 1993Serra et al , 1994Serra et al , 1997Serra et al , 2004Walter et al 1994;Morse and Draper 1995;Wu et al 1995;Laing and Hall 1996;McDowell and Turner 1996;McDowell et al 1997;Schroeder et al 1996Schroeder et al , 2003Xia et al 1997Xia et al , 1998Giese et al 1998;Kierzek et al 1999;Meroueh and Chow 1999;Dale et al 2000;Turner 2000, 2001;Burkard et al 2001;Diamond et al 2001;Mathews and Turner 2002;Proctor et al 2002;Znosko et al 2002Znosko et al , 2004Chen et al 2004Chen et al , 2005Mathews et al 2004;Vecenie and Serra 2004;Bourdélat-Parks and Wartell 2005;…”

Thermodynamic characterization of naturally occurring RNA tetraloops

“…The first set of data in Table 1 lists frequency and percent occurrence when the tetraloop nucleotides and the closing base pair are specified. Because the stability of tetraloops depends on both the identity of the nucleotides in the loop and the closing base pair Tuerk et al 1988;Antao et al 1991;Heus and Pardi 1991;Varani et al 1991;Antao and Tinoco 1992;Serra et al 1997;Giese et al 1998;Dale et al 2000;Williams and Hall 2000;Proctor et al 2002), this categorization is most important. Categorizing tetraloops in this fashion results in 524 types of tetraloops in the database.…”

“…RNA tetraloops have been extensively studied structurally (Cheong et al 1990;Heus and Pardi 1991;Varani et al 1991;Allain and Varani 1995;Jucker and Pardi 1995;Cate et al 1996;Jucker et al 1996;Ennifar et al 2000;Wimberly et al 2000) and thermodynamically Tuerk et al 1988;Antao et al 1991;Heus and Pardi 1991;Varani et al 1991;Antao and Tinoco 1992;Serra et al 1997;Giese et al 1998;Dale et al 2000;Williams and Hall 2000;Proctor et al 2002), especially the stable tetraloops 59-GNRA-39 and 59-UNCG-39. Nevertheless, less than one-third of the 30 most frequently occurring RNA tetraloops (when considering the identity of the nucleotides in the hairpin loop as well as the closing base pair) have been thermodynamically characterized.…”