Slow Conformational Dynamics at C2′-endo Nucleotides in RNA

Abstract

Local and global dynamics in folded RNAs occur over broad timescales spanning picoseconds to minutes. 1 Slow motions likely play predominant roles in governing RNA folding and ribonucleoprotein assembly reactions. However, slow local motions are extremely difficult to detect, especially for large RNAs with complex structures.The local environment and degree of flexibility can be evaluated at nucleotide resolution for RNAs of any size using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry. 2 RNA nucleotides exist in equilibrium between constrained (closed) and flexible (open) states. The 2′-OH group in flexible nucleotides preferentially adopts an open, reactive, conformation that facilitates reaction with electrophilic reagents to form a 2′-O-adduct (Figure 1). SHAPE experiments work well using electrophiles based on the isatoic anhydride (IA) scaffold. 2a,3 Positions that form 2′-O-adducts are detected by primer extension. 2 IA derivatives both react with the RNA 2′-OH group and also undergo concurrent degradation by hydrolysis (Figure 1). 2′-OH reactivity is thus conveniently monitored by allowing a reaction to proceed until the reagent has been consumed, either by hydrolysis or reaction with RNA. At this end point, the fraction adduct at any nucleotide (f) is (1) Where (2) and the rate of hydrolysis has been shown to be proportional to the rate of adduct formation, 2b,3 k adduct /k hydrolysis = β. These relationships lead to two limits. In limit 1, k open + k close ≫ k adduct [reagent] Correspondence to: Kevin M. Weeks, weeks@unc.edu. Supporting Information Available: Methods and four figures. This material is available free of charge via the Internet at http:// pubs.acs.org. HHS Public AccessIt should therefore be possible to monitor local nucleotide dynamics in RNA under conditions where limit 2 applies by varying the reactivity (or k hydrolysis ) of the hydroxylselective electrophile. IA has a hydrolysis half-life (t 1/2 ) of 430 s at 37 °C. Electronwithdrawing substituents at the cyclic amine (R 1 ) or in the benzene ring (R 2 ) enhance reagent reactivity. Compared to IA, N-methyl isatoic anhydride (NMIA), 4-nitroisatioc anhydride (4NIA), and 1-methyl 7-nitroisatoic anhydride (1M7) 3 have progressively shorter hydrolysis half-lives (table, Figure 1).To investigate if distinct local nucleotide dynamics can be captured by varying the SHAPE electrophile, we focused on an important variation in RNA structure: the C2′-endo conformation. Although C2′-endo nucleotides are relatively rare, they are highly overrepresented in important RNA tertiary interactions and in catalytic active sites. 4 Local structure at tandem G•A mismatches depends on the local sequence context. 5 Guanosine nucleotides in G•A pairs adopt the C2′-endo conformation in the sequences (UGAA) 2 5a and (GGAU) 2 , 5b the C3′-endo conformation typical of standard A-form helix geometry in (CGAG) 2 , 5c and a mixture of C2′-endo/C3′-endo conformations in (UGAG) 2 . 5b We constructed a simple hairpin RNA (termed...

C2′-endo nucleotides as molecular timers suggested by the folding of an RNA domain

“…One critical and conserved tertiary interaction in the specificity domain is a stacking interaction between A130 and A230 (in the Bacillus subtilis numbering) (21,22). A130 forms the C2Ј-endo conformation (17) and, together with its stacking partner at A230, are the only two nucleotides in the B-type RNase P specificity domain shown to be directly involved in recognizing and binding the tRNA substrate (23-25) (in red, Fig. 1A).…”

“…Slow conformational dynamics at individual nucleotides can be measured in favorable cases using SHAPE (selective 2Ј-hydroxyl acylation analyzed by primer extension) experiments that measure the differential reactivity of the ribose 2Ј-hydroxyl group toward fast versus slow-reacting reagents (17). SHAPE reagents selectively form 2Ј-O-adducts at flexible nucleotides in RNA (26,27).…”

“…1B). This strong differential reactivity with the slowly reacting reagent indicates that this C2Ј-endo nucleotide undergoes an extraordinarily slow conformational change to reach the state in which the 2Ј-hydroxyl is reactive toward acylation (t 1/2 ϭ 10-100 s) (17). When a similar experiment is performed in the absence of Mg 2ϩ , where the RNA does not form a well-defined tertiary structure (20,28), the fast and slow SHAPE reagents show equivalent reactivities at A130 (red bar in bottom panel, Fig.…”

“…S1), forms the C2Ј-endo conformation (17,21), and exhibits unusual and very slow local nucleotide dynamics (Fig. 1B).…”

“…At the static structure level, the C2Ј-endo conformation induces greater helical twist in an A-form helix (14,15) and spans a much longer 5Ј-to-3Ј distance (12,16). At the level of local motion, some C2Ј-endo nucleotides exhibit extraordinarily slow conformational dynamics with half-lives on the 10-100-s timescale (17).…”