Abstract
Mycoplasma genitalium is expected to metabolize RNA using unique pathways because its minimal genome encodes very few ribonucleases. In this work, we report that the only exoribonuclease identified in M. genitalium, RNase R, is able to remove tRNA 3'-trailers and generate mature 3'-ends. Several sequence and structural features of a tRNA precursor determine its precise processing at the 3'-end by RNase R in a purified system. The aminoacyl-acceptor stem plays a major role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA by RNase R, whereas extension of the stem results in the formation of a product terminating downstream at the new mature 3'-end. In addition, the 3'-terminal CCA sequence and the discriminator residue influence the ability of RNase R to stop at the mature 3'-end. RNase R-mediated generation of the mature 3'-end prefers a sequence of RCCN at the 3' terminus of tRNA. Variations of this sequence may cause RNase R to trim further and remove terminal CA residues from the mature 3'-end. Therefore, M. genitalium RNase R can precisely remove the 3'-trailer of a tRNA precursor by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.
Highlights
Mycoplasma genitalium lacks known ribonucleases for tRNA 3Ј-processing
Upon incubation with RNase R purified from M. genitalium, a 74-nt product corresponding to the size of the mature tRNA1Gly was produced after 5 min and increased at 30 min (Fig. 1A, lanes 2 and 3)
A 72-nt product corresponding to tRNA lacking the terminal CA residues at the 3Ј-end was produced by RNase R (Fig. 1A, lanes 2 and 3), indicating that this exoribonuclease is capable of removing nucleotide residues from the mature 3Ј-end
Summary
Mycoplasma genitalium lacks known ribonucleases for tRNA 3Ј-processing. The only identified exoribonuclease, RNase R, can carry out this function. In Bacillus subtilis, pre-tRNAs with an encoded CCA sequence are matured by exonucleolytic action at the 3Ј-end, whereas CCAless tRNA precursors are cleaved by RNase Z after the discriminator base, followed by CCA addition [7] In other bacteria such as Thermotoga maritima, the 3Ј-ends of tRNA are matured by a single endoribonucleolytic cleavage after CCA by RNase Z [8]. We report that M. genitalium RNase R demonstrates novel specificity for the nucleotide sequence and structure of a pre-tRNA, enabling tRNA 3Ј-maturation by one-step exonucleolytic removal of a relatively long 3Ј-trailer This represents a unique mechanism of tRNA 3Ј-maturation by which organisms of minimal genome employ only a single exoribonuclease
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