Studies on the hammerhead RNA self-cleaving domain

Ruffner, Duane E.; Dahm, Sue Ann C; Uhlenbeck, Oike C.

doi:10.1016/0378-1119(89)90027-9

Cited by 82 publications

(61 citation statements)

References 11 publications

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“…Although the extent of the reaction was usually 10% higher, the observed rate constant of cleavage determined with this protocol were also 0.8-1 min-l (data not shown). The value of k2 = 1 min-l is similar to values determined previously for other hammerhead constructs under similar conditions (Uhlenbeck, 1987;Ruffner et al, 1989;Fedor & Uhlenbeck, 1990,1992Dahm & Uhlenbeck, 1991;Perreault et al, 1991;Pieken et al, 1991).…”

Section: Testing the Oligonucleotides For Conformationalsupporting

confidence: 87%

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

1994

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A hammerhead ribozyme (HH16) with eight potential base pairs in each of the substrate recognition helices stabilized product binding sufficiently to enable investigation of the ligation of oligonucleotides bound to the ribozyme. All individual rate constants for product association and dissociation were determined. The following conclusions were obtained for HH16 from the analysis performed at 50 mM Tris, pH 7.5, 10 mM MgC12, and 25 "C.(1) HH16 cleaves bound substrate with a rate constant of k2 = 1 min-l, similar to rate constants obtained with other hammerhead ribozymes. (2) k-2, the rate of ligation of the 5' product and 3' product to form substrate, equaled 0.008 min-I, indicating an approximately 100-fold preference for the formation of products on the ribozyme. This internal equilibrium, compared with that for the overall solution reaction, gives an effective concentration (EC) of M for the two products bound to the ribozyme. This low EC suggests that upon cleavage of S the hammerhead complex acquires a "floppiness" which provides an entropic advantage for the formation of products on the ribozyme. (3) Product and substrate association rate constants were in the range of 107-108 M-1 min-l, comparable to values determined for short helices. (4) The stabilities of ribozyme/product complexes were similar to affinities predicted from helix-coil transitions of simple R N A duplexes, providing no indication of additional tertiary interactions. The products, P1 and P2, stabilize one another 4-fold on the ribozyme. ( 5 ) The dissociation constant for the binding of the substrate to the ribozyme was estimated to be about M.These results allowed the construction of a free energy profile for the reaction of HH16, and provide a basis for future mechanistic studies.Several plant viroids and virusoids autolytically cleave at a specific phosphodiester bond (Buzayan et al., 1986a,b;Hutchins et al., 1986; Prody et al., 1986). A consensus secondary structure of approximately 55 nucleotides termed the "hammerhead" domain has been identified (Buzayan et al., 1986b; Forster & Symons, 1987a,b) which can be assembled from 2 or 3 separate RNA molecules. Thus, the self-cleaving reaction has been turned into a multiple turnover reaction, with separate oligonucleotides acting as the "ribozyme" and the "substrate" (Figure 1) (Sampson et al., 1987;Uhlenbeck, 1987;Haseloff & Gerlach, 1988;Koizumi et al., 1988 Koizumi et al., , 1989 Jeffries & Symons, 1989 p3WAACGUC>p; PI-G, GGGAACGUCG; P1-3'p, GGGAACGUC3'-p; P2, GUCGUCGC; P2-C, GUCGUCGCC; P2-p'zCp, GUCGUCGCp"Cp; S-C, GG-G AACGUCGUCGUCGCC; p3*S-C, p3WAACGUCGUCGUCGCC; S-p'zCp, GGGAACGUCGUCGUCGCpWp; S, GGGAACGUC-GUCGUCGC; p32S, p32GGGAACGUCGUCGUCGC; E, ribozyme; HH16, hammerhead cleavage motif comprised of separate ribozyme and substrate oligonucleotides ( (HH 16). Using standardized hammerhead nomenclature (Hertel et al., 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), ...

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Section: Testing the Oligonucleotides For Conformationalsupporting

confidence: 87%

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

1994

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“…For example, the conserved sequences in the consensus active site region of hammerhead ribozymes contain 5'-GA-3' residues that are adjacent to helix II of the hammerhead (see refs. [31][32][33] Turner and coworkers (34) have found that RNA oligomers containing adjacent 5'-GA-3' mismatches are more stable than those with 5'-AG-3' mismatches. In addition, the 5'-AG-3' mismatches have imino protons in the expected region while the 5'-GA-3' sequence does not.…”

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confidence: 99%

NMR and molecular modeling evidence for a G.A mismatch base pair in a purine-rich DNA duplex.

Liu

Zon

Wilson

1991

Proc. Natl. Acad. Sci. U.S.A.

151

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ABSTRACT1H NMR experiments indicate that the oligomer 5'-d(ATGAGCGAATA) forms an unusual 10-base-pair duplex with 4 G-A base pairs (underlined) and a 3' unpaired adenosine. NMR results indicate that guanosine imino protons of the G-A mismatches are not hydrogen bonded but are stacked in the helix. A G -* I substitution in either G-A base pair causes a dramatic decrease in duplex stability and indicates that hydrogen bonding of the guanosine amino group is critical. Nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) results indicate that the overall duplex conformation is in the B-family. Cross-strand NOEs in two-dimensional NOESY spectra between a mismatched AH2 and an AHl' ofthe other mismatched base pair and between a mismatched GH8 and GNH1 of the other mismatch establish a purine-purine stacking pattern, adenosine over adenosine and guanosine over guanosine, which strongly stabilizes the duplex. A computer graphics molecular model of the unusual duplex was constructed with G-A base pairs containing A-NH2 to GN3 and G-NH2 to AN7 hydrogen bonds and B-form base pairs on both sides of the G'A pairs [5'-d(ATGAGC)J. The energy-minimized duplex satisfies all experimental constraints from NOESY and COSY results. A hydrogen bond from G-NH2 of the mismatch to a phosphate oxygen is predicted.Mismatches in DNA can arise, for example, in specific structures such as telomeres (1, 2), during replication, and in genetic recombination. If not corrected, the mismatches may lead to point mutations in subsequent replication (3-7). G-A mismatches are of particular interest because they are a common structural element in RNA folding (8)(9)(10)(11). Early studies have shown that a single G&A mismatch could be readily incorporated into DNA helices and the G-A mismatch was resistant to single-strand nucleases (12, 13). In neutral aqueous buffer with the normal base tautomers, there are four types of G&A mismatch base pairs (14), and these can be grouped by imino (Fig. 1, structures A and B) or amino hydrogen bond (Fig. 1, structures C and D) pairs with respect to guanosine. Several structural studies by single-crystal x-ray diffraction and NMR methods have shown that G-A mismatches are conformationally variable. Base pairs of types A and B but not C and D have been observed (7,(15)(16)(17)(18)(19)(20).In studies of the interaction of antisense oligonucleotides with 11-base analogs of ras p21 mRNA (21), we discovered a sequence, termed 2C, 5'-d(ATGAGCGAATA), that formed an unusually stable duplex (22). Eight of 11 bases in sequence 2C are purines and, from base-pairing possibilities, an unusual duplex with four G-A mismatch base pairs was proposed for this sequence (Fig. 2a) (22). We have undertaken NMR studies to define this unusual duplex in more detail and report here the observations of G-A mismatches oftype D and unusual purine purine base stacking in a B-form duplex.MATERIALS AND METHODS Oligonucleotides were synthesized as described (22). NMR experiments were performed on a Varian...

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“…This conformation forces the ribose-phosphate backbone to make an abrupt turn as it bridges the helical stems I and III, which in turn directs the pro-R and pro-S oxygens ofthe 3'-phosphodiester toward the inward side of the hammerhead where complexation with the Mg2+ cofactor can occur. There are no x-ray studies to date, and only preliminary NMR studies have been reported (21).Studies of a series of sequence mutations of the conserved nucleotides present in the core hammerhead structure indicate that all nine of the conserved single-stranded nucleotide residues are critical for cleavage activity (7,10,11,13,22). In all cases, mutations of the nine nonhelical conserved residues did not alter the ability of the hammerhead complex to form, but cleavage activity was eliminated or rates were reduced by >1 order of magnitude.…”

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confidence: 99%

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

McLaughlin

1992

Proc. Natl. Acad. Sci. U.S.A.

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Eight modified ribozymes of 19 residues have been prepared with individual purine amino or hydroxyl groups excised. The modified ribozymes were chemically synthesized with the substitution of a single 2'-deoxyadenosine, 2'-deoxyguanosine, inosine, or purine riboside for residues G10, A"l, G3, or A". Five of the modified ribozymes cleaved the 24-mer substrate with little change in rate as monitored by simple first-order kinetics. However, deletion of the 2-amino group at G10 (replacement with inosine) or deletion of either of the 2'-hydroxyls at G10 or G13 (replacement with 2'-deoxyguanosine) resulted in ribozymes with a drastic decrease in cleavage efficiency. Increasing the concentration of the Mg2+ cofactor from 10 mM to 50 mM significantly enhanced cleavage efficiency by these three derivatives. Steady-state kinetic assays for these three ribozymes indicated that the modifications result in both an increase in K. and a decrease in kct,. These results suggest that the exocyclic amino group at G1' and the hydroxyls at G"' and G13 are important both for ribozymesubstrate binding and for the Mg2+-catalyzed cleavage reaction.RNA-processing reactions that involve the cleavage of phosphodiester bonds are critical steps for the production of many mature RNAs from corresponding precursors and appear to be important in the replication of several plant satellite RNAs (for reviews see refs. 1-3). Autolytic self-cleavage regions are present in certain virusoid RNAs and occur in a common structural domain termed a "hammerhead" (4). The consensus structure of a self-cleaving hammerhead contains 13 conserved nucleotides held together by three helical regions (4, 5). Nine of the conserved nucleotides occur in nonhelical regions and are critical for the observed cleavage reaction. Sequence variations in the helical regions can alter the rate of the reaction (6), but only the A-U and possibly the C-G base pair at the base of stem III, at the 5' side of the cleavage site, appear to be critical for activity (7). In addition to the hammerhead structural domain, a metal cofactor is required for the observed processing reaction. Mg2" is usually employed, but Mn2+ appears to function equally well in some cases (8). Cleavage occurs at the phosphodiester bond 3' to the residue located at the bend between two of the helices (this is most commonly cytidine) (6).The in vivo reaction takes place in a unimolecular complex, but cleavage can be observed in vitro from complexes formed from two (or even three) oligonucleotides (9-12), as long as the conserved nucleotide residues and the hammerhead structure are maintained. Hammerheads composed of two RNA fragments can exist in three distinct complexes differing in the location of the respective 3' and 5' termini and the hairpin loop. All three complexes exhibit the self-cleavage reaction but the rates of cleavage (as measured by tl/2 values) can differ significantly (13). The RNA cleavage reaction, catalyzed by Mg2+ (or Mn2+), proceeds as a transesterification ofthe 2'-hydroxyl with the...

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Studies on the hammerhead RNA self-cleaving domain

Cited by 82 publications

References 11 publications

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

NMR and molecular modeling evidence for a G.A mismatch base pair in a purine-rich DNA duplex.

Importance of specific purine amino and hydroxyl groups for efficient cleavage by a hammerhead ribozyme.

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