Binding sites, ribozyme

Reference 16 discusses biochemical methods to predict specific ribozyme metal-binding sites and spectroscopic methods to identify and examine specific sites. 

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). 

Fig. 3. The hepatitis delta virus ribozyme. A Secondary structure of the genomic HDV ribo-zyme RNA used for the determination of the crystal structure [37]. The color code is reflected In the three dimensional structure B of this ribozyme. PI to P4 indicate the base-paired regions. Nucleotides in small letters indicate the U1 A binding site that was engineered into the ribozyme without affecting the overall tertiary structure. The yellow region indicates close contacts between the RNA and the U1 A protein...

Fig. 11. Comparison of the peptidyl transfer reaction in the ribosome and in the selected peptidyltransferase ribozyme. The ribosome contains a binding site for the peptidyl-tRNA (P-site) and for the aminoacyl-tRNA (A-site). In the selected ribozyme the binding site for the AMP-Met-Bio substrate would be analogous to the P-site. The attacking a-amino group which is bound in the A-site in the ribosome is covalently attached to the 5 -end in the ribozyme. Catalytically active RNAs preferentially attach the biotin tag onto themselves and can thus be separated from the inactive ones...

The ribosome is both the site of protein synthesis and an active participant in the process. The eukaryotic ribosome is constructed from two subunits the smaller 40S subunit and the larger 60S subunit. Basically, the 40S subunit binds the mRNA and monitors the recognihon between the mRNA codon and tRNA anticodon. The 60S subunit has the binding sites for aminoacyl-tRNAs and catalyzes the formation of peptide bonds. Remarkably, the catalytic entity for peptide bond formahon in the 60S subunit is the RNA component, not the protein component. Therefore, the 60S subunit acts as a ribozyme. 

Horton et al. analyzed the Mn -binding properties of hammerhead ribozyme-substrate complexes by EPR [81]. The results are consistent with the two-phase folding model. They found two classes of metal-binding sites with higher affinity and lower affinity, by monitoring the number of bound Mn + ions per hammerhead ribozyme-substrate complex at various concentrations of NaCl. They observed, in the presence of a constant concentration of Mn + ions, a sudden decrease in the number of bound low-affinity Mn ions at a lower concentration of NaCl, followed by a slow decrease or a plateau value of the number of bound high-affinity Mn ions at a higher concentration of NaCl. For example, in the absence of NaCl and in the presence of either 0.3 mmol/1 or 1 mmol/1 Mn + ions, the number of bound Mn ions per hammerhead ribozyme-substrate complex was approximately 14. Addition... 

In our recent study of reaction kinetics, we observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na ions on a background of a low concentration of either Mn or Mg ions [82]. At lower concentrations of Na ions, Na ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na ions had a rescue effect. We propose that these observations can be explained if we accept the existence of two kinds of metal-binding site that have different affinities. Our data also support the two-phase folding theory [77-80, Fig. 7], in which divalent metal ions in the ri-bozyme-substrate complex have lower and higher affinities, as proposed by Lilley and coworkers on the basis of their observations of ribozyme complexes in the ground state. 

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/... 

Self-splicing KNA. The precursor to the 26S rRNA of Tetrahymena contains a 413-nucleotide intron, which was shown by Cedi and coworkers to be selfsplicing, i.e., not to require a protein catalyst for maturation.581 582 This pre-rRNA is a ribozyme with true catalytic properties (Chapter 12). It folds into a complex three-dimensional structure which provides a binding site for free guanosine whose 3-OH attacks the phosphorus at the 5 end of the intron as shown in Fig. 28-18A, step a. The reaction is a simple displacement on phosphorus, a transesterification similar to that in the first step of pancreatic ribonuclease action (Eq. 12-25). The resulting free 3-OH then attacks the phosphorus atom at the other end of the intron (step b) to accomplish the splicing and to release the intron as a linear polynucleotide. The excised intron undergoes... 

Kisseleva, N., Kraut, S., Jaschke, A., and Schiemann, O. (2007). Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing. HFSPJ. 

Schiemann, O., Fritscher, J., Kisseleva, N., Sigurdsson, S. T., and Prisner, T. F. (2003). Structural investigation of a high-affinity Mnll binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal-ion binding. ChemBioChem 4, 1057—1065. 

Finally, novel nucleic acid catalysts have also been selected from random sequence pools (reviewed in Ref. 19). Joyce and co-workers have manipulated the function of the Group I self-splicing ribozyme, selecting variants that can utilize calcium or cleave DNA from partially randomized pools [20,21], Lorsch and Szostak [22] selected a polynucleotide kinase ribozyme from a completely random sequence pool that flanked a previously selected ATP binding site. Many of the novel ribozymes can catalyze reactions that are relevant to protein biosynthesis, bolstering arguments that translation may have arisen in a putative RNA world. For example, Lohse and Szostak [23] have selected ribozymes that can carry out an acyl transfer reaction, while Illangasekare et al. [24] have isolated a... 

Specific metal ion binding sites are directly observed in the crystal structures of hammerhead ribozymes [18], P4—P6 domain of Tetrahymena group I intron [19], transfer (tRNA) [20], GAAA tetraloop receptor [21], sarcin-ricin loop [22], and MMTV pseudoknots [23], for example. High... 

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