Abstract
We developed an in vitro cleaving assay for the thymidylate synthase (td) group I intron and observed that the off-rate of the substrate is faster than cleavage. From the sequence stems P1 and P2 can vary from 4 to 8 and from 6 to 10 base pairs, respectively, with folding of a long P1 stem being in competition with folding of a long P2 stem. Shorter substrates, which cannot compete with the formation of an extended P2, result in faster cleavage, suggesting that binding of the substrate indeed interferes with folding of stem P2. In vivo splicing analyses of mutants containing alterations in stems P1 and P2 indicate that the wild-type exon sequence of P1 is suboptimal for splicing. Furthermore, folding of P1 in vivo is in competition with an alternative cryptic P1 stem resulting in mis-splicing. Translation promotes splicing at the correct 5' splice site, whereas in the absence of translation, mis-splicing is favored. The combination of the in vitro and in vivo assays clearly displays the folding problems for correct splice site selection in this group I intron.
Highlights
We developed an in vitro cleaving assay for the thymidylate synthase group I intron and observed that the off-rate of the substrate is faster than cleavage
To verify that the ribozyme td WT-12 cleaves the substrate WT-S at a single position that correlates with the natural 5Ј splice site, a cleavage experiment was performed
The product band was compared with a labeled oligonucleotide that was designed to represent the predicted product obtained after correct 5Ј SS cleavage
Summary
Transcription of the Ribozyme—The ribozyme was transcribed in vitro from PCR products amplified from plasmid td⌬P6-2 [26] with the following primers: the coding primer with the TD T7-7 (5Ј-CCA AGT AAT ACG ACT CAC TAT AGG GCC TGA GTA TAA GGT GA-3Ј, VBC-Genomics, Austria) containing the T7 promoter and the intron sequence, starting 7 nucleotides downstream of the 5Ј splice site, whereas the antisense primer TD 3Ј end-5 (5Ј-TGT TCA GAT AAG GTC GTT AAT C-3Ј, VBC-Genomics) ended 5 nucleotides upstream of the 3Ј splice site. After the addition of the labeled substrate (ϳ0.5 nM S* in 10 mM MgCl2 and 50 mM NaMOPS, pH 7.0) the reaction was incubated for 30 min at 25 °C. After this incubation time the samples were mixed with 30% glycerol and loaded immediately on a 8% nondenaturing polyacrylamide gel running at 5 W for 1 h. After the addition of splicing buffer (final concentration, 50 mM Tris-HCl, pH 7.3, 0.4 mM spermidine, 5 mM MgCl2) the RNA was incubated for 10 min at 37 °C. Quantification was done with a Molecular Dynamics PhosphorImager, and average values where calculated from three different RNA preparations
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