Folding problems of the 5' splice site containing the P1 stem of the group I thymidylate synthase intron: substrate binding inhibition in vitro and mis-splicing in vivo.
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.
Pichler
Andrea
A
Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr. Bohrgasse 9/4, A-1030 Vienna, Austria.
Schroeder
Renée
R
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D012326:RNA Splicing / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry; D013940:Thymidylate Synthase / Q000235:genetics*
D016337:RNA, Catalytic; D013940:Thymidylate Synthase; D009355:Neomycin