Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads.
Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (-) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC approximately ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.
Carbonell
Alberto
A
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos s/n, 46022 Valencia, Spain.
De la Peña
Marcos
M
Flores
Ricardo
R
Gago
Selma
S
D001483:Base Sequence; D002384:Catalysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014772:Viroids / Q000201:enzymology*
D009711:Nucleotides; D020319:Oligodeoxyribonucleotides, Antisense; D016337:RNA, Catalytic; D012367:RNA, Viral; C427204:hammerhead ribozyme; D008274:Magnesium