All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4±P6 and P3±P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3±P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3±P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles ef®-ciently catalyzed the reversal reaction of the ®rst step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the ®rst selection. The results show that the P3±P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.
In this paper we report newly selected artificial modules that enhance the k cat values comparable with or higher than those of the wild-type ribozyme with broad substrate specificity. The elements required for the catalysis of Group I intron ribozymes are concentrated in the P3-P7 domain of their core region, which consists of two conserved helical domains, P4-P6 and P3-P7. Previously, we reported the in vitro selection of artificial modules residing at the peripheral region of a mutant Group I ribozyme lacking P4-P6. We found that derivatives of the ribozyme containing the modules performed the reversal of the first step of the self-splicing reaction efficiently by using their affinity to the substrate RNA, although their k cat values and substrate specificity were uninfluenced and limited, respectively. The results show that it is possible to add a variety of new domains at the peripheral region that play a role comparable with that of the conserved P4-P6 domain.Group I intron ribozymes catalyze two consecutive transesterification reactions to excise themselves from the precursor RNAs and ligate the flanking exons together (1). Their core region consists of two completely conserved helical domains, P3-P7 and P4-P6, which are connected via base-triples, which can be seen in Fig. 1A (2-5). Biochemical studies indicate that P7 and J8/7 regions in the P3-P7 domain are essential for the catalysis of those reactions mentioned above (6 -12). A study employing the T4 td group I intron demonstrated that a mutant ribozyme lacking both the P4-P6 domain and the base triples (M1 mutant, Fig. 1B) can still perform the trans-esterification reactions with moderate activity that was 10 3 times lower than that of the wild-type ribozyme at higher concentrations of magnesium (13).Previously we performed in vitro selection by employing the M1 mutant ribozyme to obtain artificial modules that enhance the activity of the ribozyme without the conserved P4-P6 that stabilizes the active form of the ribozyme (14). Several modules were selected: L7.1, L8, or L9. The variants with a module efficiently performed the reversal reaction of the first step of self-splicing. However, the modules contained a short sequence complementary to the substrate RNA used in the selection, indicating that the role of the module was likely to assist the substrate recognition by forming extra base pairings. In fact, we confirmed that one variant was unable to perform the reaction with another substrate without the complementary sequence. Thus, it remained unclear whether it is possible to develop a true alternative module substituting the original P4-P6 domain, which does not depend on the affinity to the substrate.We report here newly selected modules that enhance the activity of the mutant ribozyme lacking P4-P6 domain with broad substrate specificity. After 12 rounds of in vitro selection followed by an additional modification and selection, the selected variants performed the reaction with second order rate constants (k cat /K m values) one order of mag...
An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP.
Edited by Christian GriesingerKeywords: RNA-protein complex Kissing loop interaction RNP nano-object a b s t r a c t Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA-protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single triangular RNP via RNA kissing loop (KL) interactions. This RNP-based approach can be used as an alternative tool to produce unique, multifunctional molecules with customized dimensions, functions, and targets.
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