Per cell, 61 tRNA types are required to provide one-to-one correspondence between tRNA molecules and codons that specify amino acids, as there are 61 sense codons of the standard genetic code.
Amino Acids Each codon codes for one amino acid. For this reason, a variety of tRNA molecules are needed in order to accommodate not only the variety of codons but also the different types of amino acids in the body. Humans typically use 20 different amino acids. Even before genome sequencing, it was already known by direct RNA sequencing that in Escherichia coli K12, the number of different tRNA species is greater than the total number of isoacceptors All together, the total number of tRNA species in E.
The genome sequence of E. In all these cases, the sequence changes perfectly maintain base pairing and the conserved structural features of tRNA. All together in E. A well-studied example in tissue-specific expression of distinct tRNAs having the same anticodon is derived from the silkworm, Bombyx mori 13 — Previous tRNA sequencing work shows that at least two distinct tRNAs with the same anticodon is expressed in humans This work describes a surprisingly large diversity of tRNA genes among 11 species of eukaryotes from budding yeast to human.
Furthermore, the number and percentage of such tRNA genes follow the phylogenetic arrangement of these 11 organisms. Our previous experience with tRNA microarrays 17 , 18 suggests that the sensitivity and reproducibility in the detection and quantification of tRNA can be significantly improved by using hybridization probes that are complementary to the entire tRNA.
Of course, the application of full-length hybridization probes does not allow the discrimination of single nucleotide differences in tRNA. Here, we describe a systematic method that utilizes the advantage of full-length hybridization but still allows discrimination of single nucleotide differences in tRNA. Following raw data collection, human tRNA sequences were manually curated to determine the extent of variation in 11 discrete tRNA regions.
Variation in stem regions was coded to indicate whether or not acceptable base pairing Watson—Crick plus G—U is maintained. Among the human tRNA sequence set, just three sequences with greater than two pairing errors were found and excluded from our analysis. A model 30mer RNA oligonucleotide for testing the ligation method was designed to contain minimal secondary structure when the nucleotide at the 15th position is U Table 1.
The ligation reaction using the purified tRNA mixture from human samples was conducted under different conditions for hybridization and ligation. The hybridization reaction containing ng total tRNA, 0. Reactions were treated with 0. There are, however, significant outliers.
We chose to focus on tRNA sequences from 11 eukaryotic genomes as they represent a wide range in the phylogenetic tree and encompass many model organisms Figure 1 and Table S1. These 11 genomes have predicted tRNA gene counts from fission yeast to worm. The number of tRNA isoacceptors among these 11 species range from 41 budding yeast to 55 chimp. A Cladogram of the organisms based on the NCBI taxonomy browser 40 , 41 which include two single cell yeast, worm, fruit fly, fugu, chicken and five mammals, dog, rat, mouse, chimp and human.
B The number of tRNA genes left , isoacceptors middle and isodecoders right in these organisms. What is remarkable and not predicted before genome sequencing, however, are the numbers of tRNA genes having the same anticodon sequence but differences elsewhere in the tRNA body Figure 1.
One tRNA sequence within each isoacceptor class, generally the one with the highest gene copy number, is arbitrarily designated as the majority member. The number of tRNA isodecoder genes within an isoacceptor class is the count of distinct tRNA sequences within this class excluding the majority member. They differ by a single nucleotide at position By this account, the total number of different tRNA gene sequences in these 11 genomes is the number of isoacceptors i.
The fraction of tRNA isodecoder genes the sum of all isodecoder genes divided by the total number of tRNA genes has distinct groupings among these 11 species when plotted on a cladogram Figure 1A.
This phylogenetic grouping indicates that the diversity of tRNA isodecoder genes cannot be simply derived from inaccuracies in genome sequencing a small number of them may be attributed to lower sequencing accuracy in some genomes. The fraction of tRNA isodecoder genes corresponding to the phylogenic grouping of these organisms may suggest that they perform some kind of heretofore under-appreciated functions. It may also be a result of genome expansion.
The number of tRNA isoacceptors range from 41 to These isoacceptors occur between 1 and 60 times in the genome Figure 2A. Budding yeast and fruit fly have relatively few tRNA genes — and the number of occurrences for each gene is relatively low. Worm has a high number of tRNA genes and the number of occurrences is broadly distributed. A few isoacceptors in mammals have high copy numbers that distinguish them from the other isoacceptors.
Gene copy numbers of tRNA isoacceptors versus the number of occurrence or the number of isodecoders. A Plot of the gene copy number of tRNA isoacceptors and the number of occurrence for each isoacceptor class. A good linear correlation R -value between 0. For the non-mammalian species, the tRNA sequence variants are most similar within the same organism. The tRNA sequence variants are conserved across mammalian species. Each phylogenetic branch has unique sequence signatures e.
The number of tRNA isodecoder genes varies from very low 10 in yeast to very high — in chimp and human. The number of tRNA isodecoder genes in mammals has a good linear correlation R -value of 0. The highest slope possible in this plot would be 1. A slope of 0. As for the non-mammal species, a linear correlation has significantly lower R -values 0.
This result suggests that the evolutionary appearance of tRNA isodecoder genes in non-mammals may be less directed than in mammals. Among the bacterial genome sequences, the number of isodecoder genes range from 0 to 26 and the fraction of tRNA isodecoder genes range from 0 to 0. A great majority of species cluster in the lower regime of the tRNA gene-isodecoder gene plot Supplementary Figure S1.
This isoacceptor is chosen on the basis of simplicity of comparison as well as the number of isodecoder genes in each species. This tRNA isoacceptor has 11 gene copies in yeast, 15 copies in worm, 8 copies in fruit fly and mouse, and 9 copies in chimp and human.
The yeast genes have 2 sequence variants, 10 being the same plus 1 distinct isodecoder gene. Worm has 3 sequence variants, 13 being the same plus 2 isodecoders. Fruit fly, mouse and chimp have two isodecoders each and human has three isodecoders. The yeast tRNA sequences are noticeably different from all others. All tRNA genes in worm and fruit fly are clustered together among themselves. The mammalian sequences cluster more closely according to their isodecoder genes than to their species.
In fact, the majority sequence with six copies each of these mammalian species is identical. Sequence change in one worm isodecoder gene is an A—U to G—U in the stem of the long variable loop. We further analyzed the locations of sequence changes in human tRNA isodecoder genes in detail Figure 4.
The internal promoters constitute two discrete regions corresponding to nt 8—19 box A and 52—62 box B of a tRNA. Human tRNA genes vary at 6. These sequence differences may lead to differential tRNA expression in human tissues or developmental stages. Frequency of human isodecoder gene variations. Percentages indicate observed changes in each region divided by the total number of nucleotides assessed in that region. B Percent sequence variations in nine regions according to the tRNA secondary structure.
Invariant and anticodon nucleotides in all tRNAs are shown as filled black or gray circles. Percentages in parentheses stems only indicate sequence changes that result in non-Watson—Crick and non-GU base pairs. The number of sequence changes is determined by comparison of isodecoders to the majority variant. Frequency of sequence changes is the number of changes in each of the nine regions divided by the total number of nucleotides surveyed in that region.
Therefore, The next most variable region is the D-loop These high frequency regions overlap with the A and B boxes that constitute the internal promoters for Pol-III transcription. Sequence changes in the stems are between 2. More than four-fifths of sequence changes in the stems follow the rules of Watson—Crick base pairing and G—U wobble.
The function of tRNA isodecoders containing A—C pairs may depend on local pH which can vary among subcellular environments. Experimental methods used to analyze the expression of RNA transcripts are generally based on hybridization differences of complementary oligonucleotide probes, primer extension using a mixture of deoxy and dideoxynucleotide triphosphates, and RT—PCR using primers that allow differential extension by reverse transcriptase.
These methods work well when the RNA transcript is not very structured and post-transcriptional modifications do not impede hybridization or extension by the reverse transcriptase.
Although HeLa tRNAs can sometimes be detected by at least one of these methods, the result was either poorly reproducible or had very low sensitivity data not shown. We devised a systematic method to distinguish tRNA isodecoder products that differ by a single nucleotide. There are four nucleotides in DNA: adenine, guanine, cytosine and thymine. These nucleotides, also known as bases, are arranged in sets of three called codons. Some codons code for the same amino acid, and so the actual number of tRNA molecules needed is less than This redundancy in the genetic code is referred to as "wobble.
Each codon codes for one amino acid. It is the function of tRNA molecules to translate the genetic code from bases into amino acids. For this reason, a variety of tRNA molecules are needed in order to accommodate not only the variety of codons but also the different types of amino acids in the body.
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