A Novel Endonucleolytic Mechanism to Generate the CCA 3′ Termini of tRNA Molecules in Thermotoga maritima

Asako Minagawa,Hiroaki Takaku,Masamichi Takagi,Masayuki Nashimoto

doi:10.1074/jbc.m313951200

Asako Minagawa, Hiroaki Takaku + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m313951200

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Abstract

The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.

Highlights

Every single tRNA molecule ends with the sequence CCA (1)
The CCA sequences of all Escherichia coli tRNAs are encoded in its genome, and the six exoribonucleases RNase BN, RNase II, polynucleotide phosphorylase, RNase PH, RNase D, and RNase T are involved in the removal of 3Ј trailers to generate the CCA termini (17, 18)
In the course of compilation of tRNA, 3Ј tRNase, and exoribonuclease genes from available prokaryote genome data, we became aware that the Thermotoga maritima genome (19) encodes orthologues to 3Ј tRNase, RNase II, and polynucleotide phosphorylase but no orthologues to RNase BN, RNase PH, RNase D, and RNase T, its 46 tRNA genes encode CCA with only one exception (Table I)

Summary

EXPERIMENTAL PROCEDURES

Construction of Expression Plasmids for 3Ј tRNases from T. maritima, Thermoplasma acidophilum, and Pyrobaculum aerophilum— Twelve DNA fragments were chemically synthesized to produce the double-stranded DNA encoding T. maritima 3Ј tRNase, in which the codons are optimized for translation in E. coli. The full-length DNA for 3Ј tRNase (data not shown) was created by PCR with the primer pair

The abbreviations used are

A New Mechanism to Generate the tRNA Terminal CCA

RESULTS

DISCUSSION