Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity.
Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.
De la Peña
Marcos
M
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, Valencia 46022, Spain.
Gago
Selma
S
Flores
Ricardo
R
D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic; C427204:hammerhead ribozyme