Ribozymes analysis

In mid-1997 an international conference took place in Santa Cruz, USA, in which, for the first time, the exclusive topic was structural aspects of RNA molecules. A report covering this meeting contains an impressive graphic which shows the RNA structures, RNA/DNA complexes, and RNA/protein complexes contained in the brookhaven database as a function of the year of their publication [29]. Between 1988 and 1993 there were just 20. However, in 1996 alone no less than 41 structures appeared. These new dimensions were headed by the crystal structural elucidation of the first larger RNA molecule since the first crystal structure of tRNA in 1973 [30], the 48 nucleotide long hammerhead ribo-zyme (HHR) [31-33]. This landmark achievement was followed by a crystal structure analysis of the P4-P6-domain of a group I intron [34-36] and, more recently, a crystal structure of the hepatitis delta virus ribozyme [37]. 

In 1994 and 1995, two crystal structures of hammerhead ribozymes [31,32] and a structural analysis based on fluorescence resonance energy transfer studies [41] were published. In case of the crystal structure analyses, both ribozyme variants contained certain modifications that had been introduced to avoid self-cleavage [31,32]. In one case a DNA-analog of the substrate oligonucleotide was used [31], in the other case the all-RNA substrate contained a I -O-CR modification at the attacking 2 -OH group to avoid cleavage in the crystal [32] for reviews see [8,42,43]. 

Burke JM, Butcher SE, SargueU B (1996). Structural analysis and modifications of the hairpin ribozyme, p 129-144. In Eckstein F, Lilley DMJ (ed) Catalytic RNA, vol. 10. Springer, Berlin Heidelberg New York... 

This inhibitory effect of NaCl can be explained on the basis of the data from Horton et al. [81] that is described above. The Na ions remove Mn ions from the lower affinity site(s), which is somehow involved in the ribozyme activity, from the ribozyme-substrate complex. When Hammann et al. used ions in their NMR analysis, they noticed that the apparent Ka for the lower affinity ions depended on the concentration of Na ions [80]. An increase in the background concentration of NaCl from 10 mmol/1 to 50 mmol/1 weakened the affinity of the Mg ions for the complex. Their observations also reconcile with the observed inhibition by Na ions in our study, with the competitive removal of Mg /Mn ions from the ribozyme-substrate complex. 

In studies of the reactions mediated by the ribozyme from the Tetrahymena group I intron, detailed kinetic and thermodynamic analysis, combined with modifications at the atomic level, helped to define the reaction mechanism of this ribozyme at the atomic level [27, 48, 123-128]. Modification at the atomic level has generally involved replacement by a sulfur atom of an... 

It is clear that the analysis of thio effects, rescue experiments and other experiments with derivatives have contributed significantly to our understanding of the mechanism of the action of the large group I intron ribozyme of Tetmhymena. All the available data appear to support the Lewis acid catalysis for activation of the attacking nucleophile and enhancement of the leaving group that is shown in Fig. IIB. 

Lehman, N. Joyce, G.F. (1993). Evolution in vitro Analysis of a lineage of ribozymes. Current Biology... 

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 7.5 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.

Figure 14.2. RNA fold to form secondary structure. The nucleotide sequence of ribozyme (B chain of URX057 from NDB) is submitted to RNA mfold server for fold analysis. The output includes computed structure (as shown) and thermodynamic data in text (as shown) and dot plot (not shown).

Deduce the probable conformations for the two chains of a hammerhead ribozyme with the following sequences by performing folding analysis, geometry optimization and dynamic simulation (heating to and equilibrate at 300 K for 5 ps). 

Fig. 5.3.4. Mutation analysis of the tripartite ribozyme version (absolutely conserved nucleotides are in black and underlined). The activity profile is shown on a logarithmic scale.

Fig. 5.3.6. HPLC analysis of Diels-Alder reactions catalyzed by 49nt d-RNA and the enantiomeric 49nt L-ribozyme. Samples were analyzed on a chiral column with UV detection...

Another top-down approach, namely phylogenetic analysis of contemporary protein enzymes is also of limited help. Very early primordial systems must have been enzyme-free, and later early systems could have been catalysed by ribozymes. Consequently, a substantial part of basic metabolism could have originated in an era about which there simply cannot be memories in protein coding genes. 

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