Escherichia coli protein StpA stimulates self-splicing by promoting RNA assembly in vitro.
An Escherichia coli gene, stpA, has been identified and cloned based on its ability to suppress the Td- phenotype of a resident, splicing-defective phage T4 td (thymidylate synthase) gene. The stpA gene, which was localized to 60.24 min on the E. coli chromosome, encodes a 15.3-kDa protein. Overproduction of StpA in vivo led to an increase in td pre-mRNA levels and modest enhancement of td mRNA:pre-mRNA ratios. Consistent with its in vivo effect, purified StpA promoted RNA splicing in vitro, and facilitated RNA annealing and strand exchange with model substrates. These results suggest that StpA promotes splicing of the intron by binding RNA nonspecifically, resolving misfolded precursor molecules and facilitating association of critical base pair elements. Furthermore, proteinase K treatment of StpA-assembled precursors prior to the initiation of the splicing reaction still resulted in splicing enhancement, indicating that StpA is not required for the catalytic step, unlike the Neurospora splicing effector CYT-18, whose presence was necessary for catalysis to proceed. Together these results suggest that StpA has chaperone activity in vitro, with the property of promoting assembly of the precursors into an active conformation, in contrast to splicing effectors that stabilize the catalytically active intron structure.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics; D007438:Introns; D008957:Models, Genetic; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D013940:Thymidylate Synthase / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D018832:Molecular Chaperones; D012322:RNA Precursors; C090104:StpA protein, E coli; D012313:RNA; D013940:Thymidylate Synthase