Phosphates scissile phosphate

The first published crystal structure of the full length HHR [126] in which there was no solvent or ions resolved showed A9 and the scissile phosphate in close proximity, consistent with the interpretation of thio effect measurements [130], and the G8 02 and G12 Ni poised to act as a general acid and base, respectively, as proved in previous photocrosslinking [131] and mutation experiments [132], Given the strong evidence that Mg2+ participates directly in the catalytic process together with the spatial proximity of the A9 and scissile phosphate, made the placement of an Mg2+ ion in bridging position a reasonable assumption. 

Tlie two proposed metal binding sites were (1) the ribozyme P9/G10.1 site, located at the junction between stem (helix) II and the conserved catalytic core, and (2) the substrate Pl.l, scissile phosphate, site (see Figure 6.18). 

It has been proposed that the metal ion also has an interaction with the pro-Rp oxygen atom at the scissile phosphate, Pi.i. However, the scissile phosphodiester bond, within the crystal structure, is located approximately 20 A from the A9/G10.1 site [96]. It should be true that a conformationd change occurs to rotate the initial structure that is not feasible for the inhne attack to in-line structure around the scissile phosphate, but molecular dynamics analysis denies to arrange A9/G10.1 to come close to Pi.i via a metal ion. Furthermore, although it has been suggested that there is a weak interaction of a metal ion with the oxygen atom at P 1.1, our recent experiments observed no interaction of a metal ion with the Pi.i phosphate by kinetic and NMR analyses [97, 98]. 

Figure 11.3 Parallel-eye stereoscopic image of a model of the complete VS ribozyme (Lipfert et al., 2008). The model was constructed by connecting previously defined helical sections of a low-resolution model fitting the density map shown in Fig 11.2. Energy-minimization refinement against the standard stereochemical restraints was used to regularize and refine the structure. The scissile phosphate is shown as a sphere, and the probable active site components A756 and G638 are annotated.

RNase P. RNase P is a trans-acting ribozyme (150-500 nucleotides) that processes pre-tRNAs by endonucleolytically cleaving off leader sequences from the 5 -end. RNase P positions and activates a water molecule to attack the scissile phosphate. 

In group I introns, the attacking nucleophile in the first step is the 3 -OH group of an external guanosine (Fig. Ic). In the second step of splicing, the 5 -exon terminus attacks the 3 -scissile phosphate, which results in spliced exons and a linear intron with a 5 -terminal guanosine. 

During splicing, the two metal ions form additional contacts to the substrate. Evident from phosphorothioate substitution experiments are coordinative bonds to the pro-Rp oxygens of the scissile phosphates at both splice sites and to the 3 -oxygens of the leaving groups in both steps of splicing (Fig. 4). 

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