The Hammerhead Ribozyme

The hammerhead ribozyme, so named because of its claw hammer shape, is a small catalytic motif conserved in plant viroids. Viroids are infectious agents 

The hammerhead motif has a conserved secondary structure consisting of a three-way helical junction. The helical elements may vary in base sequence among species but thirteen bases at the three-way helical junction are conserved and essential for ribozyme activity. X-ray structures to be discussed below define a domain organization based on the tertiary folding observed in 

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. 

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  

Enzyme residues in bold and larger font size. 

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HammerheadRtbozyme. A small RNA molecule that catalyzes cleavage of the phosphodiester backbone of RNA is known as the hammerhead ribozyme. This ribozyme occurs namrally in certain vimses where it facihtates a site-specific self-cleavage at the phosphate and generates a 2 3 -cychc phosphate and a 5 -hydroxyl terminus. The reaction requires a divalent metal ion, such as or, as a cofactor. Whereas the... 

Fig. 10. Three-dimensional stmcmre of the hammerhead ribozyme (shaded cord) bound to a substrate oligonucleotide (nonshaded cord). The uridine mm...

Figure 14-8. The 3D density contour maps (yellow) of Na+ ion distributions derived from the activated precursor simulation. The hammerhead ribozyme is shown in blue with the active site in red. Only the high-density contour is shown here to indicate the electrostatic recruiting pocket formed in the active site...

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

The hammerhead ribozyme and leadzyme belong to the second class of ribozymes. The short extra sequences of the ribozymes form the so-called catalytic loop which acts as the enzyme. There are two likely functions for metal ions in the mechanism of action of hammerhead ribozymes formation of metal hydroxide groups or direct coordination to phosphoryl oxygens. 

Herschlag s group continued its study of structure-function relationships in the hammerhead ribozyme using a base-rescue biochemical method. This method substitutes other atoms or molecules for bases at critical catalytic or structural positions and tests whether catalytic activity is lost. If so, the RNA bases (U, A, G, C) or a modified base (for instance, deazaguanine or 2-aminopurine substituted for guanine) is added to the solution to ascertain... 

Figure 6.13 Structure of the hammerhead ribozyme HH16. Dashed boxes surround the 13 substituted positions.

Additional interactions and rearrangements in the transition state with other rescuing bases may take place because it is known from crystal structures that substrate atoms are not in line for nucleophilic attack in the hammerhead ribozyme (at least not in the published crystal structures). Also, a metal ion located -20 A away from the catalytic site was shown to be crucial for catalysis. This same metal ion appeared likely to take on an additional ligand in the transition state, suggesting that conformational changes had to take place before catalysis. ... 

Figure 6.17 Talo-5 C-methyl substituent at the hammerhead ribozyme active site.

Blount and Uhlenbeck believe that answers may be found to the above-described inconsistencies for the hammerhead ribozyme by studying naturally occurring hammerheads in which the closing loops of stems I and II interact with each other to produce a much more compact and closely packed hammerhead active conformation. The tertiary interaction between the stems appears to lower the Mg + concentration required for full catalytic activity. The reference 61 and 63 authors believe that further study of the naturally occurring hammerheads that exhibit close conformational interactions between domains I and II may lead to a more unifying view of the catalytic cleavage mechanism. 

Fig.l. The Hammerhead ribozyme. A Sequence and secondary structure. B Three dimensional structure of the HHR according to Scott et al. [33]. The substrate oligonucleotide (blue) is hybridized to the catalytic part (cyan)... 

Thomson JB, Tuschl T, Eckstein F (1996). The Hammerhead ribozyme, p 173-196. In Eckstein F, Lilley DMJ (ed) CataJytic RNA, vol 10. Springer, Berlin Heidelberg New York... 

Fig. 1A-F The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in black. The substrate or exon portion is printed in gray. Arrows indicate sites of cleavage by ribozymes A (left) the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis right) The y-shaped structure of the hammerhead ribozyme-sub-strate complex B-F the two-dimensional structures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from S. cer-evisiae (aiy5), and the ribozyme of RNase P from E. coli...

Fig. 4A The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid-base catalysts. B The single-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. One metal ion binds directly to the pro-Rp oxygen and functions as a general base catalyst. C The double-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. Two metal ions bind directly to the 2 -oxygen and the 5 -oxygen...

Fig. 6A,B Titration with ions. The hammerhead ribozyme reaction was examined on a background of ions A data obtained by Lott et al. [54]. The proposed binding of metal ions is illustrated B data obtained by Nakamatsu et al. [87]. An unmodified ribozyme (R34 gray curve) and a modified ribozyme (7-deaza-R34 black curve) were used. The rate constants were normalized by reference to the maximum rate constant ([La ]=3 (mol/1). Reactions were performed under single-turnover conditions in the presence of 80 nmol/1 ribozyme and 40 nmol/1 substrate at 37 °C...

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]...

Fig. 8 Effects of the monovalent Na" cation on the hammerhead ribozyme reaction in the presence of the divalent cation Mn. The reactions were performed under singleturnover conditions ([Ribozyme]>>[Substrate]). They were 15 min-reaction in the presence of 1 mmol/1 Mn ions at various concentrations of Na ions from 0 to 3000 mmol/ 1 or no Mn ions at 3000 mmol/1 Na ions...

The A9/G10.1 site in its conserved core region of the ribozyme has been well identified as a metal binding site by many researchers. This is strongly supported or confirmed by kinetic, NMR and X-ray crystal analyses [83-95]. The important metal-binding site within the hammerhead ribozyme, A9/... 

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