Tetrahymena ribozyme

Cech s group was the first to have success in this direction (Piccirilli, 1992). Using a genetically modified Tetrahymena ribozyme, they were able to hydrolyse an ester bond between the amino acid A-formylmethionine and the corresponding tRNAf Met. The reaction was, however, very slow, only about 5 to 15 times faster than the uncatalysed reaction. The authors ventured to suggest that these ribozymes could have functioned as the first aminoacyl tRNA synthetases. 

Tetrahydropyridines, 21 111 Tetrahydroquinolines, 21 187,192,198-199 Tetrahydroxyalkylethylenediamine titanate complexes, 25 95 Tetrahydroxyborate anion, 4 256-258 Tetrahydroxycuprate, 7 770 Tetrahymena, ribozyme from, 17 618, 619 Tetrairidium dodecacarbonyl, 16 63 Tetraisopropyl titanate, 25 92 Tetrakaidecahedron structure, 12 8 Tetrakis(2,4-di-lerl-butylphenyl)-4,4 -biphenylenediphosphonite, 3 114 Tetrakis(2-chloroethyl)... 

Figure 6.5 Triple base pair for the Tetrahymena ribozyme (PDB 1X8W). Visualized using CambridgeSoft Chem3D Ultra 10.0 with notations in ChemDraw Ultra 10.0. (Printed with permission of CambridgeSoft Corporation.)...

Figure 2. The catalytic reaction cycle of the Tetrahymena ribozyme (E), showing the binding and docking reactions (leading to the formation of E-S complex), followed by a bond cleavage breaking step (the rate constant for which is kchem) and release of the 5 -fragment in the multiturnover steps (rate constant equals k t).

Uhlenbeck, O.C. (1987) A small catalytic oligoribonucleotide. Nature, 328.596-600. Zaug, A.J., Michael, D.B. and Cech. T.R. (1986) The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature, 324, 429 33. 

Smaller self-cleaving RNAs have been found among plant viruses and viroids. Many of them have a common catalytic core which can be converted into 30- to 40-nucleotide ribozymes. Only 17 nucleotides and three hydrogen-bonded helical stems are required to form the self-cleaving "hammerhead" domain, which has a structural similarity to the catalytic core of the Tetrahymena ribozyme. The hammerhead ribozymes (Fig. 12-27) represent one form of small ribozyme.793-797 Another is the hairpin ribozyme shown in Fig. 

Figure 12-26 (A,B) Stereoscopic views of the Tetrahymena ribozyme. (A) Ribbon tracing of the phosphodiester backbone.

Strobel, S. A., and Cech, T. R. (1993). Tertiary interactions with the internal guide sequence mediate docking of the PI helix into the catalytic core of the Tetrahymena ribozyme. Biochemistry 32, 13593-13604. 

To resolve folded and unfolded conformations of the 387 nt Tetrahymena ribozyme, we use 8% acrylamide (29 1 mono bisacrylamide) in 34 mMTris, 66 mM Hepes (pH 7.5), 0.1 mM EDTA, and 3 mM MgCl2 (THEM3). Hepes is used instead of borate to maintain the native structure of the ribozyme (Buchmueller and Weeks, 2004 Pyle et ah, 1990), whereas the MgCl2 concentration is chosen to be just sufficient to maintain the RNA in its folded state. Twenty-five milliliters of acrylamide solution per gel is prepared and degassed using RNase-free water. 200 /iL 10% ammonium... 

For the folding reactions (5-10 iL), labeled ribozyme (1000-2000 cpm/ fiV) is added to HE buffer (50 mM Hepes adjusted to pH 7.5 with sodium hydroxide, 1 mMEDTA, pH 8), 10% (v/v) glycerol, 0.01% (w/v) xylene cyanol, plus the desired concentration of MgCl2 or other salt (Heilman-Miller et al, 2001 Koculi et ah, 2004). At least one sample should contain no MgCl2, representing the unfolded RNA, and one sample should contain enough MgCl2 to fold the RNA completely. The reactions are incubated at the desired temperature for sufficient time for the reaction to reach equilibrium. We incubate the Tetrahymena ribozyme 2-4 h in a water... 

When the folding reactions have reached equilibrium, 2 fiL of each sample is loaded into separate lanes of a native 8% polyacrylamide gel that was prepared and prerun as described above (< 10 ° C). It is helpful to use narrow tips that allow the sample to be placed near the bottom of the well. The current should be applied to the gel as soon as possible after the samples are loaded. Gels must be run long enough to resolve the conformational species of interest, but not so long that smaller RNAs run off the bottom of the gel. To resolve the folded and unfolded forms of the Tetrahymena ribozyme, gels are run 4 h at 15 W. 

Figure 9.4 Mobility of the Tetrahymena ribozyme in different divalent metal ions. (A) The unfolded (U) and folded (F) ribozyme was run next to OX DNA size markers on native 8% PAGE in THE buffer with 3 mM MgCl2, CaCl2, or SrCl2. (B) The relative RNA mobility decreased with the size and charge density of the metal ion. Reprinted from Koculi et al. (2007).

For example, misfolded forms of the Tetrahymena ribozyme refold very slowly at 4 °C, and are easily separated from the native form (Pan and Woodson, 1998). However, if the ribozyme is first incubated in another ion such as Na+ that allows the RNA to come close to the native structure, these native-like intermediates are captured as the native form when the RNA encounters Mg2+ in the gel running buffer (Figure 9.3A) (Heilman-Miller et al, 2001). Similarly, the Azoarcus ribozyme rapidly forms nativelike, compact intermediates in Mg2+ concentrations below that required for catalytic activity (Rangan et al, 2003). These intermediates also appear in the folded state when assayed by native PAGE. 

Heilman-Miller, S. L., Thirumalai, D., and Woodson, S. A. (2001). Role of counterion condensation in folding of the Tetrahymena ribozyme. I. Equilibrium stabilization by cations. J. Mol. Biol. 306, 1157—1166. 

Ortoleva-Donnelly, L., Szewczak, A. A., Gutell, R. R., and Strobel, S. A. (1998). The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA 4, 498-519. 

Figure 12.4 The time-dependent radius of gyration for the Tetrahymena ribozyme following the addition of 10 mM Mg2+ to RNA in solutions containing either low (20 mM, brown) or moderate (100 mM, blue) concentrations of monovalent ions.

Structured RNAs that are significantly smaller than the Tetrahymena ribozyme can be studied in the same way as a model oligonucleotide... 

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