Hammerhead RNA

McKay and co-workers determined the X-ray crystallographic structure of a hammerhead RNA-DNA ribozyme complex at 2.6-A resolution in 1994, deposited in the Protein Databank (PDB) as IHMH. The substrate DNA strand ensured that the complex would remain in the ground state since the DNA 2 -deoxy position could not undergo the cyclization/cleavage reaction. 

Scott, W. G., Finch, J. T., Grenfell, R., Fogg, J., Smith, T., Gait, M. J. and Klug, A. (1995). Rapid crystalhzation of chemically synthesized hammerhead RNAs using a double screening procedure. J. Mol. Biol. 250, 327-332. 

Results are from simulations with Mg2+ initially placed at the bridging position for the reactant state (b-RT), the early transition state (b-ETS) mimic, and the late transition state (b-LTS) mimic. Average values are shown with standard deviations in the parenthesis (divided by the decimal precision). X-ray structures used for comparison include the full-length hammerhead RNA crystallographic structure at 2.2 A resolution (2GOZ) [41] that was also used in this chapter as the starting structure, and the 2.0 A resolution structure with resolved Mn2+ sites and solvent (20EU) [42]. 

Figure 5 Upper Active site of the full-length hammerhead RNA using the canonical minimal sequence numbering scheme described in [40] and [42]. Lower Representative hydrogen bonding of the C3 G8 base pair observed from mutant simulations. Experimental relative catalytic rates of mutant versus wild-type minimal sequence ribozymes (kmut/kwt) are shown in parentheses (C3U from [76], G8A from [78], C3U/G8A from [73], and G8I from [34]), and may differ for the full-length sequence.

Ruffner, D.E., Stormo, G.D., Uhlenbeck, O.C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 1990, 29,10695-712. 

Sakatsume O, Ogawa T, Hosaka H, Kawashima M, Takaki M, Takaku H. Synthesis and properties of non-hammerhead RNA using l-(2-chloroeth-oxy)ethyl group for the protection of 2 -hydroxyl function. Nucleosides Nucleotides 10 141-153, 1991. 

Similar properties are found in structure determinations of Co -soaked crystals of the hammerhead ribozyme. In the hammerhead RNA structures, Mg ions have been located in five sites, Mn ions in six sites, " and Co ions in eight sites.As described above for Mg and Mn, the coordination details of the A9/G10.1 site, which consists of phosphate/N7 ligands, changes with... 

Fig. 5-1 Ribozymes rely for their action on the great diversity of tertiary structures that RNA chains can adopt. A specific shape, so-called hammerhead RNA, has hydrolytic activity and cleaves mRNA at a specific sequence of bases.

Some nucleic acids are capable of self-splicing. These catalytic DNA and RNA are known as deoxyribozymes (Li and Breaker, 1999 Sheppard et al, 2000) and ribozymes (Doherty and Doudna, 2000 Scott and Klug, 1996) respectively. The 3 - and/or 2 -hydroxyls of DNA/RNA serve as a catalytic site that invariably requires a metal ion for the catalytic activity. Deoxyribozymes are quasi-catalytic while ribozymes can be catalytic, e.g. ribonuclease P (RNase P) as well as quasi-catalytic, e.g. introns and hammerhead RNAs. RNase P resources are maintained at http //www.mbio.ncsu.edu/RnaseP/homeJitml... 

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... 

Nash KL, Alexander GJ, Lever AM (2005) Inhibition of hepatitis B virus by lentiviral vector delivered antisense RNA and hammerhead libozymes. J Viral Hepat 12 346-356... 

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 ... 

Figure 6.10 Secondary structure of the RNA 6 hammerhead ribozyme as found in the X-ray crystallographic structures PDB IHMH, IMME, 299D, 300D, 301D, 359D, 379D, and 488D. A modified RNA6 was used for crystal structures PDB INYI, and 1Q29.

In the following year, Scott and co-workers solved the X-ray crystallographic structure of an all-RNA hammerhead ribozyme with a 2 -OCH3 group incorporated at the active site cytosine (Cn) to prevent cleavage (PDB IMME). This structure differed from that of IHMH in several important ways (1) it was an all-RNA ribozyme rather than an RNA-DNA hybrid (2) the connectivity of the ribozyme backbone strands was different (for instance... 

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... 

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