Chapter 22
Hairpin Ribozyme Structure and Dynamics 1
2
1
3
4
A. R. Banerjee , A. Berzal-Herranz , J. Bond , S. Butcher , J. A. Esteban , J. E. Heckman , B. Sargueil , N. Walter , and J. M . Burke Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch022
1
5
1
1
1
Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Burling, VT 05405
A wide range of experimental methods have been used to explore the structure and function of the hairpin ribozyme. In vitro selection methods has provided comprehensive information on RNA secondary structure and the identity of essential bases within the two internal loops of theribozyme-substratecomplex. Crosslinking analysis has identified a tertiary structure motif within ribozyme loop Β that is also found in rRNA. The ribozyme contains two domains that fold independently. A sharp bend at the junction between the two domains is necessary for the two domains to interact in the active complex. Biphasic kinetic behavior in pre-steady state cleavage kinetics results from partitioning of the ribozyme between the active, bent conformer, and an inactive conformer in which the two domains are coaxially stacked, thus preventing their interaction. Novel strategies have been used to reconstruct the ribozyme in such a way that folding into the active conformer is strongly favored. The hairpin ribozyme is a small (50 nt) catalytic R N A molecule that catalyzes a reversible RNA cleavage reaction, using a magnesium-dependent transesterification reaction which generates 5'-OH and 2\3'-cyclic phosphate termini (1-3, Gait, M.J. et al. Antisense and Nucleic Acids Drug Development, in press). In nature, the ribozyme is responsible for R N A processing reactions that are essential for replication of R N A molecules associated with plant R N A viruses. Trans-acting ribozymes have been developed through deletion of the natural sequences that are not germane to R N A processing. Such ribozymes provide excellent model systems for studying the biochemistry of R N A processing, and biological 2
Current address: Instituto de Parasitologia y Biomedicinia, CSIC, Ventanilla 11, 18001 Granada, Spain
3
Current address: Molecular Biology Institute, University of California at Los Angeles, East Los Angeles, CA 90095-1570
5
Current address: Centre National de la Recherche Scientifique, CGM, Avenue de Laterasse, 91190 Gif-Sur-Yvette, France
360
© 1998 American Chemical Society
Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
22.
BANERJEE ET AL.
Hairpin Ribozyme Structure and Dynamics
361
catalysis by RNA. Because catalytic activity is a direct consequence of the folded structure of RNA, ribozymes are excellent model systems for studying R N A structure and dynamics.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch022
Results In vitro selection. To elucidate the secondary structure of the ribozymesubstrate complex, and to identify essential bases, we developed a rapid and powerful in vitro selection method (4). This method essentially permits investigators to do both genetics and molecular evolution in the test tube, by selectively replicating molecules with user-defined catalytic properties from highly complex populations (> 10 ) of variants generated through chemical synthesis. Results define a secondary structure consisting of four short helical elements consisting of canonical base pairs (Figure 1) (Gait, M.J. et al. Antisense and Nucleic Acids Drug Development, in press). Two internal loops are present, one of which contains the substrate cleavage site. Interestingly, the identity of nearly all bases within the loops is important for activity; single base substitutions at each of these sites strongly inhibit catalysis (5-7). 12
Photoreactive motif within internal loop Β is shared with viroids and rRNA. A covalent crosslink between G21 and U42 is induced with high efficiency upon modest U V irradiation of the unmodified R N A (8). It is likely that the photoreactive species reflects the active conformation of the molecule, because structural modifications that increase photocrosslinking efficiency to greater than 90% are accompanied by a corresponding increase in catalytic activity. Similar high-efficiency crosslinks are found in R N A loops within viroids, and within 5S and 28S rRNA. In all cases, the loops have strikingly similar sequences and apparently utilize identical crosslinking sites. Structural analysis by N M R spectroscopy shows that the structures of the two rRNA loops are essentially identical; both structures include noncanonical base pairing and a local reversal of chain direction lead to base stacking across the loop; this cross-strand stacking leads to the UV-induced crosslink (9-10). Chemical modification analysis of the hairpin ribozyme is entirely consistent with that expected on the basis of the rRNA N M R structures. Therefore, we have modeled the three-dimensional structure of the relevant sequences within loop Β by homology to the r R N A models. The structure of the segment of loop Β between the photoreactive motif and helix 4 is unknown. Escape from conformational hell. R N A molecules in general, and ribozymes in particular, are notorious for the formation of multiple conformations, usually one active species and numerous inactive conformers. This makes both the prediction and experimental determination of biologically-relevant three dimensional structures very difficult. We devoted considerable effort to the identification of specific misfolded structures and to redesign of the ribozyme so as to minimize their formation. Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch022
362
MOLECULAR MODELING OF NUCLEIC ACIDS
Figure 1. Secondary structure of the complex between the hairpin ribozyme and its cognate substrate. Ribozyme and substrate sequences are in upper case and lower-case, respectively. Ribozyme nucleotides are numbered from 1 to 50. Substrate nucleotides are numbered with negative numbers 5' to the cleavage site, and vice versa. A short arrow indicates the cleavage-ligation site. The four helical domains are designated as H1-H4, and the two internal loops as A and B. Proposed non-canonical base pairs at the top of loop Β (13) are indicated. Domain A is defined as the helical element comprising H I , loop A and H2. Similarly, domain Β stands for the H3-loop B-H4 element. Three base pairs (one in HI and two in H2) of the naturally occurring sequences have been changed to minimize self-complementarity of the substrate (6, 9, 11). A rate-enhancing U39C mutation was also introduced. The putative hinge region is indicated. Inset. Schematic cartoon showing the two alternative conformations of the hairpin ribozyme. See text for details.
Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch022
22. BANERJEE E T AL.
Hairpin Ribozyme Structure and Dynamics
363
Specific problems with the wild type ribozyme were identified and corrected. First, the wild type substrate was found to assume a hairpin structure at low R N A concentrations, and to form dimers at higher concentrations. We solved this problem by redesigning the substrate and the ribozyme's substratebinding domain so as to prevent this misfolding (Heckman, J.E. et al., University of Vermont, unpublished data). Second, helix 4 of the ribozyme is metastable. Consequently, the ribozyme can dimerize even at relatively low concentrations, through the formation of intermolecular versions of domain Β (8). Although these dimers are catalytically active, we chose to engineer modified ribozymes that have increased activity and do not dimerize, through stabilization of helix 4(11). In vitro selection experiments showed that the mutation U39C was recovered as a second-site revertant of several partially inactivating mutations within the ribozyme. This mutation is a general activating mutation that may assist in formation of the active structure within loop B. C39 is now routinely incorporated into all ribozymes in our lab. Two independently-folding domains. A variety of methods were applied to examine the structure of intact hairpin ribozymes, ribozyme-substrate complexes, and ribozyme derivatives in which some sequences were deleted. Crosslinking studies, gel mobility analysis, and chemical modification experiments suggested that the ribozyme consists of two independently-folding domains. Domain A is a duplex between the substrate and substrate-binding strand (helix 1, loop A and helix 2), while domain Β consists of helix 3, loop Β and helix 4. Studies in which the effect of inserting variable-length linkers between the 5' end of the substrate and the 3' end of the ribozyme showed that coaxial stacking of helices 2 and 3 prevented intramolecular cleavage activity, and strongly suggested that a sharp bend must occur between these helices to form the active configuration (12-13). We were able to experimentally demonstrate a functional interaction between the two domains, through reconstitution of cleavage activity (14). Success of this experiment depended on having previously solved the misfolding problems described above. The separated domains are able to associate in solution to assemble a catalytically-proficient complex. The observed rate of the reaction approaches & of the unmodified ribozyme, but only at very high R N A concentrations. This demonstrates the presence of tertiary interactions between the two domains, but suggests that they are quite weak. The identity of these interactions has not yet been determined. cat
Pre-steady state kinetic analysis. To increase the depth of our understanding of the steps involved in the catalytic cycle, we have recently completed a comprehensive analysis of the rates of individual steps of the cleavage and ligation reaction pathway (15). In addition to utilizing the well-established kinetic methods for ribozymes that involve quantitative analysis of reactions using radiolabeled substrates, we developed real-time assays using fluorescent substrates and substrate analogs (16). These assays monitor quenching and Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
364
MOLECULAR MODELING OF NUCLEIC ACIDS
dequenching of 3'-fluorescein labeled substrates and analogs upon binding and dissociation, as well as loss of a fluorescence energy transfer signal upon cleavage of substrate containing both 3'-fluorescein and 5'-hexachlorofluorescein labels. Our pre-steady state kinetic analysis was conducted with at 25°C in a simple buffer containing 12 m M magnesium ions, and yielded very interesting results (75), several of which have been independently determined (77) (Figure 2). Binding of substrate is very fast (4 χ 10 M " min" ), essentially the same rate as that observed for the diffusion-limited formation of an analogous A - R N A duplex. In contrast, substrate dissociation is very slow (
