Coaxial stacking

Group I intron phosphotransesterification reactions are carried out by a conserved active site that contains a set of imperfect double helices named PI through P9. (See Figure 6.4.) P1-P9 helices are organized into three domains P1-P2, P4-P6, and P3-P9. Specifically, the Tetrahymena thermophila intron contains two sets of coaxially stacked helices that overlap to create the active site. These helices reside in two domains of approximately equal size P4-P6 and P3-P9. P domains are defined as base-paired regions, whereas J domains... 

Fig. 7 The two-stage folding scheme for the hammerhead ribozyme, as proposed by Tilley s group [77-80]. The arrow indicates the cleavage site. The scheme consists of two steps to generate the Y- or y-shaped ribozyme/substrate complex. The higher affinity of Mg is related to formation of domain II (structural scaffold non-Watson-Crick pairings between G12-A9, Ais-Gg and A14-U7 forming a coaxial stack between hehces II and III that runs through G12A13A14) and the lower affinity of Mg to formation of domain I (catalytic domain formation by the sequence C3U4G5A6 and the C17 with the rotation of helix I around into the same quadrant as helix II) [78]...

Comparative gel electrophoresis analysis of the 4H junction of U1 snRNA showed the junction adopted a coaxially stacked structure with almost perpendicular axes (Fig. 7.3). This result was very recently confirmed crystallographically (Pomeranz-Krummel et al., 2009). Perhaps the most extensively studied 4H junction in RNA is that of the hairpin ribozyme,... 

Figure 7.3 Analysis of the 4H four-way RNA junction of the human U1 snRNA by comparative gel electrophoresis (Duckett et al., 1995). The central sequence of the junction is shown. The A G pair at the center was retained in this analysis, although changing it to a Watson—Crick pair did not alter the global shape of the junction. The six long—short species can be considered to be derived from a junction with four arms of 40 bp. The central 20 bp comprises RNA, and the outer arms are DNA. The junction species were electrophoresed in an 8% polyacrylamide gel, in 90 mM Tris—borate (pH 8.3) and 1 mM Mg2+. The mobility pattern of the six species is slow, slow, fast, fast, slow, slow. The simplest interpretation (shown on the right-hand side) is that of a stacked structure based on A on D and B on C coaxial stacking, with the axes nearly perpendicular. The pattern would also be consistent with a rapid exchange between nearly equal populations of parallel and antiparallel forms. However, a recent crystal structure has found a perpendicular stacked structure for this RNA junction (Pomeranz-Krummel et al., 2009).

Figure 7.5 Analysis of the two three-way RNA junctions of the VS ribozyme by comparative gel electrophoresis. The secondary structure of the VS ribozyme is shown, with the sequences of the two component three-way junctions. Each was analyzed in isolation by comparative gel electrophoresis, comparing the mobilities of the three long-short arm species. As before, these species have a central core of RNA that is extended with DNA sections. The junction species were electrophoresed in 10% polyacrylamide gels in the presence of 90 mM Tris—borate (pH 8.3) with 3 (junction III—IV—V) or 5 (junction II—III—VI) mM Mg2. The structural interpretations of both sets of data are shown. Both junctions undergo coaxial stacking of two arms, with die third directed laterally.

In recent years further novel classes of compounds were added to cyclophane chemistry. The multilayeredphanesiS derived from [2.2]paracyclophane contain coaxially stacked benzene rings connected by ethano bridges para to one another. The first members of this series were described in 196416. Quadruple layered phane hydrocarbons 3 and 4 reveal in their UV-spectra long range electronic effects penetrating several arene units. 

DeRose and coworkers have explored conformational changes of TAR RNA upon binding of divalent metal ions (Ca " ) by measuring the dipolar coupling between two attached spin labels 20 using CW EPR (Fig. 2). The U25-U40 distances obtained from Fourier deconvolution methods are 11.9 0.3 A for TAR RNA in the absence of divalent metal cations and 14.2 0.3 A when 50 mM Ca " was added [45]. These results are in accordance with the proposed coaxial stacking of the two TAR helices upon addition of metal ions based on the X-ray crystal structure [80]. 

Helical stacking Helical stacking is one strategy for packing RNA helices into a tertiary structure. The secondary structure of tRNA consists of four short helices that radiate from the center in a cloverleaf-like shape. In its three-dimensional structure, two pairs of helices coaxially stack and perpendicularly align to yield the L-shaped tertiary structure. Coaxial stacking of helices is observed in ribozymes leading to extensive tertiary interactions between helical subdomains. 

In contrast to and Rb", lithium picrate crystallizes with torand 1 as a 2 2 complex containing three water molecules.The two torands are coaxially stacked and threaded by a hydrated dilithium chain H20-Li -H20-Li -H20. Each lithium cation binds unsyinmetri-cally to two of the six nitrogens in each torand, and two water molecules complete the tetrahedral Li" coordination sphere. No anion coordination is observed, and hydrogen... 

The algorithm incorporates recently determined thermodynamic parameters for the free energies of internal loops of 2 by 1 and 2 by 2 nucleotides. New free energy bonuses for tetraloops and triloops have been developed by consideration of the database of phylogenetically determined structures. Finally, new rules for coaxial stacking have been applied. This new version will be available in FORTRAN for Unix machines and a C++ version is now available for use on Personal Computers with Windows 95 or Windows NT. The program was used to explore structures predicted to have a free energy near the minimum. 

This preliminary report presents recent progress in secondary structure prediction based on free energy minimization. The following changes have been implemented The method for forcing base pairs has been improved. A filter that removes isolated Watson-Crick or G-U base pairs (those that cannot stack on any other Watson-Crick or G-U pair) has been incorporated. Recently measured free energies for 2 by 2 internal loops (Xia, T. McDowell, J. A. Turner, D. H. In preparation.), 2 by 1 internal loops (75), and hairpin loops 18) have also been incorporated. Finally, a new model for coaxial stacking of helixes has been developed. 

Figure 1. Coaxial Stacking of two helixes with an intervening mismatch. Stack 1 is the stack of the mismatch with a continuous backbone. Stack 2 is the stack of the mismatch with an open backbone.

Exterior Loops. Exterior loops are open loops that contain the ends of a sequence. This version of the algorithm gives bonuses for dangling ends and coaxial stacking in exterior loops using the same model as multibranch loops. 

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). 

In three dimensions, tRNAs fold into an L-shaped structure in which the acceptor stem and T C arm coaxially stack to form one part of the L known as the minihelix, and the D and anticodon arms likewise stack to form the other part of the molecule. This structure is facilitated and stabilized by tertiary interactions at the corner of the L that bring together the D and variable loops. The nucleotides involved in these interactions are typically invariant or semi-invariant, indicating that the tRNA L shape is universal. While most base pairs in tRNA helices are canonical Watson-Crick pairs, the tertiary interactions at the corner of the L make use of some unusual hydrogen-bonding conformations. For example, nearly all tRNAs contain a U8 A14 reverse Hoogsteen base pair, and several base triples (where three bases are paired together) are also typically present at the core of the structure. 

As discussed earlier, AARSs have core catalytic domains that perform the functions of aminoacyl adenylate formation and transfer of the amino acid to the cognate tRNA. The sequences and structures of these domains also differentiate the enzymes as belonging to Class I or II. In addition to this class-defining active site domain, most AARSs also have one or more appended domains that are unique. These idiosyncratic domains often make specific contacts with recognition elements outside the tRNA acceptor stem, for example, at the anticodon or variable loop of the tRNA molecule (Fig. 4). In addition to the two-domain (or more) organization of the AARS enzymes, tRNAs can also be viewed as modular structures. As mentioned earlier, the acceptor stem and T4 C arm coaxially stack to form one portion of the L-shaped tRNA structure, while the D and anticodon arms stack to make the other tRNA arm (Fig. 2). The acceptor arm makes contacts with the catalytic core of the enzyme and contains the amino acid attachment site, while the anticodon, located on the second arm of the tRNA, is recognized by an appended domain. 

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