Biomedical subjects

Tan Inoue

Publications and source records attributed to Tan Inoue.

24 records · Page 2Linked to original sources

PubMed2002

Design, construction, and analysis of a novel class of self-folding RNA.

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.

Animals↗

PubMed2002

Solution structure of an RNA fragment with the P7/P9.0 region and the 3'-terminal guanosine of the tetrahymena group I intron.

In the second step of the two consecutive transesterifications of the self-splicing reaction of the group I intron, the conserved guanosine at the 3' terminus of the intron (omegaG) binds to the guanosine-binding site (GBS) in the intron. In the present study, we designed a 22-nt model RNA (GBS/omegaG) including the GBS and omegaG from the Tetrahymena group I intron, and determined the solution structure by NMR methods. In this structure, omegaG is recognized by the formation of a base triple with the G264 x C311 base pair, and this recognition is stabilized by the stacking interaction between omegaG and C262. The bulged structure at A263 causes a large helical twist angle (40 +/- 80) between the G264 x C311 and C262 x G312 base pairs. We named this type of binding pocket with a bulge and a large twist, formed on the major groove, a "Bulge-and-Twist" (BT) pocket. With another twist angle between the C262 x G312 and G413 x C313 base pairs (45 +/- 100), the axis of GBS/omegaG is kinked at the GBS region. This kinked axis superimposes well on that of the corresponding region in the structure model built on a 5.0 A resolution electron density map (Golden et al., Science, 1998, 282:345-358). This compact structure of the GBS is also consistent with previous biochemical studies on group I introns. The BT pockets are also found in the arginine-binding site of the HIV-TAR RNA, and within the 16S rRNA and the 23S rRNA.

Animals↗

PubMed2002

Mispaired P3 region in the hierarchical folding pathway of the Tetrahymena ribozyme.

BACKGROUND: The Tetrahymena group I ribozyme folds into a complex three-dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4-P6 and P3-P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate. RESULTS: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt-P3) appears a likely candidate. To examine the effects of the alt-P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3-P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt-P3 structure were altered systematically. CONCLUSION: The results indicate that the alt-P3 structure is not the major rate-limiting factor in the folding process.

Animals↗

PubMed2002

Two conserved structural components, A-rich bulge and P4 XJ6/7 base-triples, in activating the group I ribozymes.

BACKGROUND: The A-rich bulge of the group I intron ribozyme, a highly conserved structural element in its P5 peripheral region, plays a significant role in activating the ribozyme. The bulge has been known to interact with the P4 stem forming P4 XJ6/7 base-triples in the conserved core. The base-triples by themselves have also been identified as a distinctive element responsible for enhancing the activity of the ribozyme. RESULTS: A weakly active variant of the Tetrahymena ribozyme lacking the P5 extension was dramatically activated by the addition of an A-rich bulge at the peripheral region, or by replacement of the original P4 XJ6/7 base-triples in the core structure with more stabilized isosteric ones. Biochemical analyses showed that the two methods of activation affect the ribozyme differently. CONCLUSIONS: The long-range interaction between the A-rich bulge and P4 or additionally stabilized P4 XJ6/7 base-triples can contribute dramatically to activation of the Tetrahymena ribozyme. Both improve the kcat value, which represents the rate of the limiting step of the ribozyme reaction when its binding site is saturated with GTP. However, the bulge or the modified base-triples gave a moderate reduction or considerable increase, respectively, to the Km(GTP) value.

Adenosine↗

PubMed2002

Modeling of a possible evolutional process from a ribozyme to a catalytic RNP.

A model process for molecular evolution from an RNA enzyme to a catalytic RNA-protein complex (RNP) is proposed. In the model, one RNA-RNA interaction in the enzyme is replaced by an RNA-protein interaction via an intermediary state where the original RNA-RNA and newly introduced RNA-protein interaction co-exist. To test the model, a catalytic RNP was designed and examined by employing the Tetrahymena ribozyme.

Animals↗

PubMed2002

Role of a conserved J8/7 X P4 base-triple in the Tetrahymena ribozyme.

The Tetrahymena group I intron ribozyme folds into a complex three dimensional structure for performing the self-splicing reaction. Catalysis depends on its core structure comprising two helical domains, P4-P6 and P3-P7. The two domains are joined by three sets of conserved base-triple(s) and other tertiary interactions. We found that the disruption of J8/7 X P4, one such conserved base-triple, causes the catalytic ability to deteriorate without altering the folding rate. This suggests that the base-triple stabilizes the active structure of the ribozyme but plays no significant role in RNA folding. By combining the present and previous results, it can be concluded that three sets of conserved base-triples play distinct roles in the Tetrahymena ribozyme.

Animals↗