BACKGROUND: The A-rich bulge of the group I intron ribozyme, a highly conserved structural element in its P5 peripheral region, plays a significant role in activating the ribozyme. The bulge has been known to interact with the P4 stem forming P4 XJ6/7 base-triples in the conserved core. The base-triples by themselves have also been identified as a distinctive element responsible for enhancing the activity of the ribozyme. RESULTS: A weakly active variant of the Tetrahymena ribozyme lacking the P5 extension was dramatically activated by the addition of an A-rich bulge at the peripheral region, or by replacement of the original P4 XJ6/7 base-triples in the core structure with more stabilized isosteric ones. Biochemical analyses showed that the two methods of activation affect the ribozyme differently. CONCLUSIONS: The long-range interaction between the A-rich bulge and P4 or additionally stabilized P4 XJ6/7 base-triples can contribute dramatically to activation of the Tetrahymena ribozyme. Both improve the kcat value, which represents the rate of the limiting step of the ribozyme reaction when its binding site is saturated with GTP. However, the bulge or the modified base-triples gave a moderate reduction or considerable increase, respectively, to the Km(GTP) value.
BACKGROUND: The A-rich bulge of the group I intron ribozyme, a highly conserved structural element in its P5 peripheral region, plays a significant role in activating the ribozyme. The bulge has been known to interact with the P4 stem forming P4 XJ6/7 base-triples in the conserved core. The base-triples by themselves have also been identified as a distinctive element responsible for enhancing the activity of the ribozyme. RESULTS: A weakly active variant of the Tetrahymena ribozyme lacking the P5 extension was dramatically activated by the addition of an A-rich bulge at the peripheral region, or by replacement of the original P4 XJ6/7 base-triples in the core structure with more stabilized isosteric ones. Biochemical analyses showed that the two methods of activation affect the ribozyme differently. CONCLUSIONS: The long-range interaction between the A-rich bulge and P4 or additionally stabilized P4 XJ6/7 base-triples can contribute dramatically to activation of the Tetrahymena ribozyme. Both improve the kcat value, which represents the rate of the limiting step of the ribozyme reaction when its binding site is saturated with GTP. However, the bulge or the modified base-triples gave a moderate reduction or considerable increase, respectively, to the Km(GTP) value.
Authors: Steven W Sowa; Jorge Vazquez-Anderson; Chelsea A Clark; Ricardo De La Peña; Kaitlin Dunn; Emily K Fung; Mark J Khoury; Lydia M Contreras Journal: Nucleic Acids Res Date: 2014-11-21 Impact factor: 16.971