Construction and processing of transfer RNA precursor models

C. K. Surratt, Z. Lesnikowski, A. L. Schifman, F. J. Schmidt, S. M. Hecht

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Abstract

Several 'dimeric' tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P. Construction was effected by the T4 RNA ligand-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNA(Phe) (donor substrate), followed by annealing of the 3'-half of yeast tRNA(Phe) (nucleotides 38-76). E. coli RNase P and yeast M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E. coli tRNA(Phe) (5'-tRNA) and yeast tRNA(Phe) (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNA(Phe), or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E. coli tRNA(Phe) on a polyacrylamide gel. By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage. The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al. (McClain, W.H., Guerrier-Takada, C., and Altman, S. (1987) Science 238, 527-530) that identifies the T stem and loop as possible recognition site.

Original languageEnglish (US)
Pages (from-to)22506-22512
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number36
StatePublished - Dec 1 1990

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Surratt, C. K., Lesnikowski, Z., Schifman, A. L., Schmidt, F. J., & Hecht, S. M. (1990). Construction and processing of transfer RNA precursor models. Journal of Biological Chemistry, 265(36), 22506-22512.