Pairwise alignments were made between the plasmids and the tRNA sequences to identify the att sites.
Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNAPyl was the most efficient and orthogonal tRNA.
The tRNA sequences were predicted by comparing nucleotide homology with tRNAscan-SE2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/).
If the tRNA sequence was random, one would expect better alignments than with its expected tRNA homologue for half the tRNAs with non-cognates, hence 9.5 among 19.
These results were also confirmed by the association between the pathogenic effects of human polymorphisms in tRNA genes and the cloverleaf formation of the antisense tRNA sequences [ 25].
In our data set, we found that more than half of the species (365 species) contain at least one species-specific repeat which is located prior to, following to, or between RNA gene sequences (e.g. between 23S rRNA and 5S rRNA gene sequences, between different tRNA sequences, etc).
Interestingly, the csRNAs derived from most of the chloroplast tRNA sequences (except tRNA-Ser, tRNA-Thr and tRNA-Trp) exhibited an abundant peak in the first 5% block, which accounted for more than 95% of the tRNA-derived csRNAs.
The split tRNA sequences were taken from Randau [30].
Interestingly, the maturation of a polycistronic pre-mRNA in bilaterian mitochondria involves the excision of tRNA sequences located between most coding sequences in the genome, resulting in monocistronic mRNAs (Ojala et al. 1981).
Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs.
