UC Irvine

Investigating the influence of peripheral domains on the kinetic rates of HDV-like ribozyme self-cleavage is important to discovering their biological relevance. The following work investigates the effects of peripheral elements on the self-scission of two HDV-like ribozymes, the mouse CPEB3 ribozyme and the mosquito drz-Agam-2-1.

The CPEB3 ribozyme, located in an intron of the mammalian CPEB3 gene, is the first HDV-like ribozyme discovered in a genome other than the virus. ESTs from human cells suggest ribozyme activity in vivo and demonstrate tissue-specific expression1; the level of activity in human brain tissue suggests another factor, such as a chaperone, increases ribozyme activity by minimizing misfolding2. The rates of the co-transcriptional self-scission of the CPEB3 ribozyme were determined using sequences varying by the number of nucleotides surrounding the ribozyme. Experimental rates range from 0.13 ± 0.029 to 0.46 ± 0.020 min-1 with the order of the constructs being -233/72, -197/72, -197/72/21, -233/72, -197/72/165, - 233/72/165, -10/72, -49/72/165 and -257/72/328. The findings here demonstrate that CPEB3 ribozymes are kinetically fast-reacting and suggest a relationship between flanking sequences and rate.

The drz-Agam-2-1 ribozyme was the first naturally occurring ribozyme to contain a helix in the J1/2 domain3, a domain shown to indirectly effect ribozyme self-cleaving4. The rate of self-cleavage increased with step-wise reintroduction of the helix into drz-Agam-2-1. Experiments correlated the stability derived from J1/2 to catalysis and provide an explanation for the ribozyme's Hill coefficient of n=1. The data shows the J1/2 structured element to impact ribozyme catalysis through stabilization of the catalytic core.