Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs

Abstract

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.

In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

“…Previous data have shown that the enzymatic extract used in these experiments is able (i) to correctly aminoacylate plant cytosolic tRNA Ala transcript (9) and (ii) to efficiently replace the yeast cytosolic S-100 supernatant required for import of tRNA on May 12, 2018 by guest http://mcb.asm.org/ conditions we used for in vitro tRNA import are very similar to those described for in vitro protein import into isolated potato mitochondria (51), and as a matter of fact, in vitro protein import was efficient under these tRNA import conditions (data not shown), in case coimport would have to occur. It should also be noted that preaminoacylation of tRNA Ala did not improve its import efficiency in our in vitro system (data not shown), whereas aminoacylation is an essential requirement for import of tRNA Lys into S. cerevisiae mitochondria (48).…”

“…The corresponding constructs containing A. thaliana cytosolic tRNA Ala or tRNA Phe gene sequences were obtained previously (9). The construct encoding mature A. thaliana tRNA Met-e was amplified by PCR with the relevant primers so that the intronless tRNA gene sequence was directly fused to the T7 RNA polymerase promoter at the 5Ј end and to a BstNI site at the 3Ј end (39).…”

A Single Editing Event Is a Prerequisite for Efficient Processing of Potato Mitochondrial Phenylalanine tRNA

“…In vitro runoff transcripts were prepared from these constructs as described by Perret et al (31) after digestion with BstNI. Aminoacylation was conducted under optimal conditions (8) in the presence of a potato mitochondrion or an Escherichia coli enzymatic extract (24). cDNA synthesis and amplification by PCR.…”

The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

“…AlaRS apparently primarily recognizes the conserved G3:U70 wobble base pair in the acceptor stem (Hou and Schimmel, 1988;' To whom correspondence should be addressed Forsyth et al, 1991;Gabriel et al, 1996). It has been shown that mutation of to C70 abolishes aminoacylation of tRNAAIa by AlaRS in a wide range of organisms (Hou and Schimmel, 1989), including plants (Carneiro et al, 1994). The simplicity and high conservation of this motif make it an ideal model for studying aminoacyl-tRNA synthetase-tRNA interactions.…”