Hammerhead ribozyme constructs

Figure 6.12 Secondary structure of hammerhead ribozyme constructs used for biochemical, spectroscopic, and kinetic studies. (A) the HHal ribozyme and (B) the HH8 ribozyme. (Reprinted with permission from Figure IB of reference 47.)...

Figure 6.18 Rescue sites in the HH16 hammerhead ribozyme construct.

Many researchers refer to stems 1, 2, and 3 using their Roman numeral equivalents—that is, stems I, II, and III. These motifs are also denoted as helices I, II, and III. It should be noted at the beginning of this hammerhead ribozyme discussion that structure-function relationships, the role of various nucleobases, metal ion participation in catalysis, and other features of the system have not been completely delineated and in some cases remain controversial. Globally, the hammerhead fold appears to be similar in both solution and solid-state studies. In solution, however, the central core of the hammerhead construct appears to be highly dynamic. This may account for different experimental results among the analytical techniques used in solution and certainly explains some distinct differences seen between solution and solid-state (X-ray crystallographic) structures. 

First identified in 1986 as the catalytic active element in the replication cycle of certain viruses, the hammerhead ribozymes (HHRz) are the smallest known, naturally occurring RNA endonucleases They consist of a single RNA motif which catalyzes a reversible, site-specific cleavage of one of its own phosphodiester bonds . Truncation of this motif allowed a minimal HHRz to be constructed which was the very first ribozyme to be crystallized. HHRz minimal motifs are characterized by a core of eleven conserved nucleotides (bold font in Figure 20) from which three helices of variable length radiate. Selective mutation of any of these conserved residues results in a substantial loss of activity. In the absence of metal ions the structure is relaxed ( extended ), but upon addition of Mg +, hammerhead ribozymes spontaneously fold into a Y-shaped conformation (Figure 20 Color Plate 3). ... 

The implication of the hammerhead structures is that, because the base sequences of helices 1 and 111 are not conserved, a hammerhead ribozyme can be constructed from completely unrelated RNAs and perform rapid, site-specific cleavage reactions in trans on virtually any target sequence. This versatility, combined with the structural simplicity and portability, makes the hammerhead ribo-... 

Hammerhead construct RNA 6 16 nt ribozyme, 25 nt substrate. Used for Scott group crystal structures and biochemical experiments by Hampel and Burke, references 33, 56 5 3 ... 

Hampel and Burke observed that protection of hammerhead backbone sites in Mg + solutions required assembly of the full ribozyme-substrate complex. In other words, testing of ribozyme or substrate separately in the hydroxyl footprinting assay showed essentially complete hydrolysis of all nucleotides (Figure 2B of reference 56). In contrast, the fully assembled ribozyme-substrate complex showed protection of nucleotides structurally near the densely packed three-helix junction of hammerhead constructs HH16, HHal, and RNA 6. Two of the ribozyme group of protected nucleotides (Gs, Ae) are part of the conserved uridine U-turn seen in all known hammerhead constructs. (See Figures 6.10,6.11, and 6.12.) The footprinting results are collected in Table 6.5. 

Hammerhead Construction Ribozyme Strand Substrate Strand Additional Ribozyme Site... 

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