A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme

Abstract

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

Figure 1

The Tetrahymena group I ribozyme and its J1/2 junction shown schematically. (A) The P1 duplex (green) docks into the catalytic core (blue); K_dock (= [docked]/[undocked]) is the equilibrium constant between the undocked and docked states. The single-stranded J1/2 junction (red) connects the substrate containing P1 duplex to the P2 duplex (beige), one of the several peripheral segments of the ribozyme. P denotes paired regions, and J denotes junctions that connect the paired regions. (B) J1/2 (red) and its surroundings, with individual residues highlighted that are mutated or discussed herein. (C) Schematic model of the J1/2 (red line) conformation in the docked state based on results herein. J1/2 exits P2 and is bent to allow the center A of J1/2, A29 (shown in red), to form a tertiary interaction involving the first base pair in P2, A31•U56 (blue); see also Figure S3. The catalytic cleavage site is located a specified number of residues 3′ of this interaction (see Table 1 & text).

Figure 2

The effect of J1/2 base sequence on (A) the docking equilibrium constant, K_dock, and (B) catalytic activity k_obs. The four sequences with the center residue (position 29) as A (blue) are represented by the blue line, and the four sequences with A29 replaced by U (red) are represented by the red line. K_dock in part (A) was obtained from smFRET (see Supporting Information). Activity in part (B) was measured by following the reaction of (E•S•G)_o using a ³²P-radiolabeled open complex substrate under single-turnover conditions with saturating enzyme and saturating G (2 mM) that assay both docking and the chemical step.

Figure 3

Testing a potential contact between A29 and two base pairs in P2, A31•U56 (A) and G32•C55 (B) via double mutant cycles. The wild-type sequence has A29, A31•U56, and G32•C55. The introduced mutations are shown in bold. The numbers adjacent to the arrowed lines are the fold decrease in k_obs from introduction of the mutation. The amounts of coupling between the A29 interaction and the P2 mutation are shown on the right.

Figure 4

Co-conservation of A29 and the A31•U56 base pair. The height of the letter for a base type i, i = A/U/G/C, at residue r in the sequence logos is f_i,r*I_r in unit of bits, where f_i,r is the fraction of residue r that is base type i and I_r = 2 + sum(f_i,r*log₂f_i,r) is the information content of residue r. The top sequence logo includes all sequences that have a residue 29. (Residue 29 is defined as the second base 5′ to P2 in the single stranded region of J1/2; sequences with J1/2 of 2 nt or longer all have a residue 29; see also Tables 1 and S3.) The middle sequence logo includes only the sequences with A at residue 29. The bottom sequence logo includes the sequences with residue 29 being anything other than A. The dots under residue 28-30 indicate unpaired residues and the brackets under residue 31 and 56 indicate base pairing.