The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme

Abstract

Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an a-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 39-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 29-fluoro A, but not with 29-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C29-endo conformation. In other cases where there is interference with 29-deoxy A, the presence or absence of 29-fluoro A interference helps to establish whether the 29-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.

An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site

“…The unreactive fractions of all mutant ribozymes were equally small (Ͻ15%), eliminating misfolding as a potential cause for the difference in reaction rates. To further confirm that k cat is This set of mutations restores the predicted base triple with an isomorphous triple that is the next most phylogenetically conserved sequence in the IC1 and IC2 intron subgroups (11). In every case that U300 is changed to a C within these subgroups, the A97-U277 base pair co-varies as a G-C pair.…”

“…Wild-type and mutant L-21 G414 and L-21 ScaI ribozyme RNAs were synthesized by T7 RNA polymerase transcription of EarI-and ScaI-digested plasmid DNA, respectively, and were purified on denaturing polyacrylamide gels (11). For NAIM and NAIS experiments, A␣S, 7dA␣S, m 6 A␣S, G␣S, 7dG␣S, C␣S, and dC␣S were randomly incorporated at low levels (Յ5%) into wild-type and mutant ribozymes as described (11).…”

“…Recently, the results of NAIM and NAIS experiments to identify the array of hydrogen bonding interactions that stabilize the interaction between the P1 substrate helix and the highly conserved J8͞7 strand within the intron core were reported (7,11). These data, along with other biochemical structural information (4-6, 12, 13), were used as restraints in building a model of a large section of the ribozyme active site.…”

“…At the 5Ј end of J8͞7, phylogenetic evidence suggests that U300 may form a triple with the A97-U277 base pair in the P3 helix (Figs. 1 and 2) (11,16), although this has not been tested biochemically.…”

Local RNA structural changes induced by crystallization are revealed by SHAPE

“…By offering a biochemical signature of C29-endo sugar puckers, nucleotide analog interference mapping using 29-fluoro and 29-deoxy derivatives could provide that distinction (Ortoleva-Donnelly et al 1998).…”