Five steps in the conversion of a large precursor RNA into bacteriophage proline and serine transfer RNAs

Abstract

We describe the nucleotide sequence derivation of the precursor RNA to bacteriophage T4 proline and serine transfer RNAs. Large oligonucleotide fragments obtained by limited digestion of the precursor RNA define the arrangement within the precursor RNA as 5′-proline tRNA-serine tRNA-3′. The nucleotide sequences of the transfer RNAs are also derived, in the Appendix for proline tRNA and in the accompanying paper by McClain et al. (1975) for serine tRNA. Comparisons of nucleotide sequences show that the precursor RNA lacks 3′ CCAOH residues of both transfer RNA species, indicating that these residues must be added enzymatically during precursor RNA maturation. The precursor RNA to bacteriophage T2 proline and serine tRNAs is nearly identical in nucleotide sequence to the T4 precursor RNA. The only difference is at the 3′ terminus where the CCAOH residues of serine tRNA are present in the T2 precursor RNA and are believed to be synthesized during transcription. We have characterized the enzymatic steps involved in the conversion of precursor RNA into proline and serine tRNAs. The studies rely on nucleotide sequence determinations of incompletely matured precursor RNAs that accumulate in Escherichia coli cells deficient in the enzymes associated with precursor RNA maturation. The combined results of previous and present studies permit us to propose the following pathway for the appearance of the transfer RNAs. The process is initiated by the addition of CCAOH to the 3′ terminus of the T4 precursor RNA through the combined action of an exonuclease and tRNA nucleotidyltransferase. Both enzymes are of host origin. The T4 precursor RNA intermediate generated by these reactions is identical to the T2 precursor RNA. T2 and T4 precursor RNAs terminating in CCAOH are then cleaved twice by the host enzyme ribonuclease P to yield two transfer RNA-size products that contain the 5′ termini of the transfer RNA species. The cleavage product derived from the 3′ end of the precursor RNA and terminating in CCAOH is serine tRNA; addition of a 2′-O-methyl group to this transfer RNA species represents the terminal step in the biosynthesis of serine tRNA. The second ribonuclease P cleavage product is a species of immature proline tRNA that lacks its 3′ CCAOH residues. Proline tRNA is then formed by the combined action of the exonuclease and tRNA nucleotidyltransferase.