A Rearrangement of the Guanosine-Binding Site Establishes an Extended Network of Functional Interactions in the Tetrahymena Group I Ribozyme Active Site

Abstract

Protein enzymes appear to use extensive packing and hydrogen-bonding interactions to precisely position catalytic groups within active sites. Due to their inherent backbone flexibility and limited side chain repertoire, RNA enzymes face additional challenges relative to proteins in precisely positioning substrates and catalytic groups. Here, we use the group I ribozyme to probe the existence, establishment, and functional consequences of an extended network of interactions in an RNA active site. The group I ribozyme catalyzes a site-specific attack of guanosine on an oligonucleotide substrate. We previously determined that the hydrogen bond between the exocyclic amino group of guanosine and the 2′-hydroxyl group at position A261 of the Tetrahymena group I ribozyme contributes to overall catalysis. We now use functional data, aided by double-mutant cycles, to probe this hydrogen bond in the individual reaction steps of the catalytic cycle. Our results indicate that this hydrogen bond is not formed upon guanosine binding to the ribozyme but instead forms at a later stage of the catalytic cycle. Formation of this hydrogen bond is correlated to other structural rearrangements in the ribozyme's active site that are promoted by docking of the oligonucleotide substrate into the ribozyme's active site, and disruption of this interaction has deleterious consequences for the chemical transformation within the ternary complex. These results, combined with earlier results, provide insight into the nature of the multiple conformational steps used by the Tetrahymena group I ribozyme to achieve its active structure and reveal an intricate, extended network of interactions that is used to establish catalytic interactions within this RNA's active site.Enzymatic reactions use the same chemical groups and functionalities that can be found on simple molecule catalysts, but the enzymatic reaction take place in specialized pockets, referred to as active sites. The protein structure provides networks of packing and hydrogen bonding interactions to precisely position substrates and catalytic groups within the active site (1). The identification and functional characterization of these networks of interactions † This work was supported by a grant from the NIH (GM 49243) to D.H. and by a grant from the Howard Hughes Medical Institute to J.A.P. *Address correspondence to this author at the Department of Biochemistry, Beckman Center, B400, Stanford University, Stanford,. herschla@stanford.edu. ⊥ Present address: Department of Biochemistry, Stanford University. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 30. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript is a fundamental step in understanding of how enzymes provide their extraordinary rate acceleration and specificity.Structural techniques, such as X-ray crystallography, are powerful tools to garner information about networks of interactions but alone are not sufficient to determine the role of specific ...

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

“…Fig. 1B,C; Shan and Herschlag 1999), and these and other data (Bevilacqua et al 1993;McConnell et al 1993;Herschlag 1999, 2002;Forconi et al 2010Forconi et al , 2011 provide strong evidence for a conformational change within the active site from E•G to E•S•G.…”

“…5A,B). Functional data (Forconi et al 2010) suggest that this hydrogen bond is made in (E•S•G) C but not (E•S•G) O and we speculate that the absence of this contact releases the adjacent C262 residue, a ligand for M C (Hougland et al 2005). Such a change might allow M C to sample alternative positions (Fig.…”

“…1A), first by base-pairing with E to form the (E•S) O complex (where "O" stands for open) and then by docking of the substrate-containing duplex, referred to as P1, into the active site via tertiary interactions to form the (E•S) C complex (where "C" stands for closed) (Bevilacqua et al 1992(Bevilacqua et al , 1994Herschlag 1992;Li et al 1995;Narlikar and Herschlag 1996). Prior results strongly suggest that the interactions made with G are the same within the E•G and (E•S•G) O complexes because S is absent from the active site in both complexes (Wang et al 1993;Narlikar and Herschlag 1996), but are different within the (E•S•G) C complex (McConnell et al 1993;Wang et al 1993;Shan and Herschlag 1999;Karbstein et al 2002;Forconi et al 2010). …”

“…We also altered the conformation of the A261 ribose ring to ensure that its 2 ′ -OH group is not donating a hydrogen bond to the G exocyclic amino group, as prior biochemical data provided evidence for the absence of this interaction (Forconi et al 2010). We then applied harmonic distance restraints based on the available functional data (Supplemental Table S1) to ensure an interaction between M A and G 3 ′ -OH (Fig.…”

“…5B) following the same procedures but with both G -OH groups contacting M C (Fig. 3C) and a hydrogen bond between the G exocyclic amino group and the A261 2 ′ -OH group (Materials and Methods; Supplemental Table S1; Forconi et al 2010).…”