Sequence and Structure Requirements for Drosophila tRNA 5′- and 3′-End Processing

Abstract

Eukaryotic tRNAs are processed at their 5'- and 3'- ends by endonucleases RNase P and 3'-tRNase, respectively. We have prepared substrates for both enzymes, separated the activities from a Drosophila extract, and designed variant tRNAs to assess the effects of sequence and structure on processing. Mutations affect these reactions in similar ways; thus, RNase P and 3'-tRNase probably require similar substrate structures to maintain the catalytic fit. RNase P is more sensitive to substrate substitutions than 3'-tRNase. In three of the four stems, one substitution prevents both processing reactions while the opposite one has less effect; anticodon stem substitutions hardly affect processing, and double substitution intended to restore base pairing also restore processing to the wild type rate. Structure probing suggests that tRNA misfolding sometimes coincides with reduced processing. In other cases, processing inhibition probably results from specific unfavorable stem appositions leading to local helix deformation. A single T loop substitution disrupts the tertiary D-T loop interaction and reduces processing. We have thus begun mapping tRNA processing determinants on the global, local, and tertiary structure levels.

Of proteins and RNA: The RNase P/MRP family

“…pre-tRNA recognition by eukaryotic RNase P Recognition of pre-tRNA by eukaryotic RNase P requires more substrate elements than in bacteria (Yuan et al 1992;Levinger et al 1995;Yuan and Altman 1995;Ziehler et al 2000). Human, Xenopus laevis, and Drosophila melanogaster RNase P can cleave minimized substrates made up FIGURE 1.…”

Naturally Occurring Mutations in Human Mitochondrial Pre-tRNASer(UCN) Can Affect the Transfer Ribonuclease Z Cleavage Site, Processing Kinetics, and Substrate Secondary Structure

“…32 P]ATP, gel-purified, and recovered as described (14). Additionally, pre-tRNA Arg was 3Ј end-labeled with ␣-32 P-labeled cyticline 3Ј-phosphate and T4 RNA ligase (31).…”

Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3′ end processing

“…Fruit fly pre-tRNA His , the tRNase Z substrate used throughout (Frendewey et al 1985;Levinger et al 1995), is a 108-nt transcript consisting of a 72-nt tRNA (to the discriminator) and a 36-nt 39-end trailer (substrate and tRNA product bands are marked on the right side of Fig. 4B).…”

“…Runoff transcription with T7 RNA polymerase using plasmid templates has been described before; tRNA His with a 36-nt 39-end trailer and mature tRNA His -CCA were designed to run off using DraI and NsiI RE ends, respectively, for use as tRNase Z substrates (Levinger et al 1995;Mohan et al 1999). The new template for tRNA His -AUG (a positive control for tRNase Z reaction designed to closely match the length of mature tRNA His ) was prepared by introducing a SacI site at the appropriate position by amplification using a mismatched 39-end primer and a universal 59-end primer.…”

“…In eukaryotes, archaea, and many bacteria in which CCA is not transcriptionally encoded-on the other hand-the CCA-adding enzyme is essential (Aebi et al 1990). In these cases, in a widely conserved, efficient, and precise mechanism for pre-tRNA 39 end maturation, a pre-tRNA 39 end processing endonuclease, tRNase Z, cleaves the 39-end trailer directly following the discriminator (the last unpaired nucleotide following the 7-bp acceptor stem), leaving a 39 OH to which CCA can be directly added (Castaño et al 1985;Frendewey et al 1985;Chen and Martin 1988;Marchfelder et al 1990;Nashimoto 1992;Levinger et al 1995; for reviews, see Levinger et al 2004b;Vogel et al 2005). Interestingly, tRNA precursors with and without transcriptionally embedded CCA coexist in Bacillus subtilis, and the two pathways for pre-tRNA 39 end maturation are used selectively on the appropriate substrates (Pellegrini et al 2003).…”