Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity.
The hammerhead ribozyme (HHRz) is a small, naturally occurring ribozyme that site-specifically cleaves RNA and has long been considered a potentially useful tool for gene silencing. The minimal conserved HHRz motif derived from natural sequences consists of three helices that intersect at a highly conserved catalytic core of 11 nucleotides. The presence of this motif is sufficient to support cleavage at high Mg2+ concentrations, but not at the low Mg2+ concentrations characteristic of intracellular environments. Here we demonstrate that natural HHRzs require the presence of additional nonconserved sequence elements outside of the conserved catalytic core to enable intracellular activity. These elements may stabilize the HHRz in a catalytically active conformation via tertiary interactions. HHRzs stabilized by these interactions cleave efficiently at physiological Mg2+ concentrations and are functional in vivo. The proposed role of these tertiary interacting motifs is supported by mutational, functional, structural and molecular modeling analysis of natural HHRzs.
Khvorova
Anastasia
A
Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA. khovorova.a@dharmacon.com
Lescoute
Aurélie
A
Westhof
Eric
E
Jayasena
Sumedha D
SD
D020816:Amino Acid Motifs; D001483:Base Sequence; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D005822:Genetic Vectors; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry*; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D012313:RNA; D008274:Magnesium