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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.

Carbonell A, De la Peña M, Flores R, Gago S - Nucleic Acids Res. (2006)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos s/n, 46022 Valencia, Spain.

ABSTRACT
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.

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Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft = F∞ (1−e−kt), where F∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F∞ (between parenthesis) are shown below. The concentration of Mg was ∼1000-fold higher than that of hammerheads.
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fig3: Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft = F∞ (1−e−kt), where F∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F∞ (between parenthesis) are shown below. The concentration of Mg was ∼1000-fold higher than that of hammerheads.

Mentions: At very low magnesium concentration (10 μM), the ELVd-(+)-GUC mutant (where the GUC refers to the trinucleotide preceding the self-cleavage site) presented a self-cleavage rate constant significantly higher than the wild-type ELVd-(+)-AUA hammerhead (Figure 3A). The ELVd-(+)-GUA mutant had a self-cleavage rate constant intermediate between those of the ELVd-(+)-GUC mutant and the ELVd-(+)-AUA wild-type but, surprisingly, the ELVd-(+)-AUC mutant displayed the highest rate constant (Figure 3A). Moreover, at the end-point of the reaction, 95% of molecules from the three mutants with the highest rate constants self-cleaved, whereas this fraction was only 60% for the wild-type ELVd-(+)-AUA hammerhead (Figure 3A). A similar trend in the rate constants was observed when the magnesium concentration was increased moderately (50 μM): ELVd-(+)-AUC≈ELVd-(+)-GUC>ELVd-(+)-GUA>ELVd-(+)-AUA, with the fraction of self-cleaved molecules at the end-point of the reaction being higher than 95% in all cases (Figure 3B). At higher magnesium concentration (500 μM), the self-cleavage rates were too fast to be analyzed manually (self-cleavage was essentially complete after 30 s, data not shown). Altogether these results indicate that the effects of changes in the trinucleotide preceding the self-cleavage site follow the same tendency observed previously in artificial trans-acting hammerheads under high magnesium concentration (38–41). However, they neither provide hints on why some catalytically sub-optimal trinucleotides like AUA have been selected in natural hammerheads, nor on why the catalytically most favorable trinucleotide AUC has been essentially excluded in natural hammerheads.


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.

Carbonell A, De la Peña M, Flores R, Gago S - Nucleic Acids Res. (2006)

Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft = F∞ (1−e−kt), where F∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F∞ (between parenthesis) are shown below. The concentration of Mg was ∼1000-fold higher than that of hammerheads.
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Related In: Results  -  Collection

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fig3: Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft = F∞ (1−e−kt), where F∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F∞ (between parenthesis) are shown below. The concentration of Mg was ∼1000-fold higher than that of hammerheads.
Mentions: At very low magnesium concentration (10 μM), the ELVd-(+)-GUC mutant (where the GUC refers to the trinucleotide preceding the self-cleavage site) presented a self-cleavage rate constant significantly higher than the wild-type ELVd-(+)-AUA hammerhead (Figure 3A). The ELVd-(+)-GUA mutant had a self-cleavage rate constant intermediate between those of the ELVd-(+)-GUC mutant and the ELVd-(+)-AUA wild-type but, surprisingly, the ELVd-(+)-AUC mutant displayed the highest rate constant (Figure 3A). Moreover, at the end-point of the reaction, 95% of molecules from the three mutants with the highest rate constants self-cleaved, whereas this fraction was only 60% for the wild-type ELVd-(+)-AUA hammerhead (Figure 3A). A similar trend in the rate constants was observed when the magnesium concentration was increased moderately (50 μM): ELVd-(+)-AUC≈ELVd-(+)-GUC>ELVd-(+)-GUA>ELVd-(+)-AUA, with the fraction of self-cleaved molecules at the end-point of the reaction being higher than 95% in all cases (Figure 3B). At higher magnesium concentration (500 μM), the self-cleavage rates were too fast to be analyzed manually (self-cleavage was essentially complete after 30 s, data not shown). Altogether these results indicate that the effects of changes in the trinucleotide preceding the self-cleavage site follow the same tendency observed previously in artificial trans-acting hammerheads under high magnesium concentration (38–41). However, they neither provide hints on why some catalytically sub-optimal trinucleotides like AUA have been selected in natural hammerheads, nor on why the catalytically most favorable trinucleotide AUC has been essentially excluded in natural hammerheads.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos s/n, 46022 Valencia, Spain.

ABSTRACT
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.

Show MeSH