Nucleoside composition of lysine transfer RNAs from Lupinus luteus seeds.

Analysis of the nucleoside composition of five lysine tRNAs from lupin seeds has shown their general similarity to other eukaryotic lysine tRNAs, except that lupin lysine tRNAs do not contain either t6A, Tm, or thioderivatives of uridine. It is assumed that each of the lupin tRNALys is coded for by a separate gene. The acceptor activity of the analysed tRNAs ranged from about 1200 (tRNA3Lys, tRNA4Lys, tRNA5Lys) to 1470 (tRNA2Lys) pmoles of lysine per one A260 unit of tRNA.     

The role of modifications in codon discrimination by tRNA(Lys)UUU

The natural modification of specific nucleosides in many tRNAs is essential during decoding of mRNA by the ribosome. For example, tRNA(Lys)(UUU) requires the modification N6-threonylcarbamoyladenosine at position 37 (t(6)A37), adjacent and 3' to the anticodon, to bind AAA in the A site of the ribosomal 30S subunit. Moreover, it can only bind both AAA and AAG lysine codons when doubly modified with t(6)A37 and either 5-methylaminomethyluridine or 2-thiouridine at the wobble position (mnm(5)U34 or s(2)U34). Here we report crystal structures of modified tRNA anticodon stem-loops bound to the 30S ribosomal subunit with lysine codons in the A site. These structures allow the rationalization of how modifications in the anticodon loop enable decoding of both lysine codons AAA and AAG.     

Lysine tRNAs from rat liver: lysine tRNA sequences are highly conserved

The two major lysine tRNAs from rat liver, tRNA2Lys and tRNA5Lys, were sequenced by rapid gel or chromatogram readout methods. The major tRNA2Lys differs from a minor form only by a base pair in positions 29 and 41; both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Although highly related, the major tRNA2Lys and tRNA5Lys differ in four base pairs and four unpaired nucleotides, including the first position of the anticodons, but have the same base pair in positions 29 and 41. The three tRNAs maintain a m2G-U pair in the acceptor stem. Detection of this m2G is in contrast to other reports of lysine tRNAs. Sequences of lysine tRNAs are strongly conserved in higher eukaryotes.     

Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.

Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.     

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.     

Calcium induction of transglutaminase and the formation of epsilon(gamma-glutamyl) lysine cross-links in cultured mouse epidermal cells

A lysine tRNA (anticodon U¹UU) was isolated from rat liver mitochondria and sequenced. The sequence, pCAUUGCGAm¹Am²GCUUAGAGCm²GUUAACCU$\text{U}{}^1\text{UU}$-t⁶AAGUUAAAGUUAGAGACAACAAAUCUCCACAAUGACCA_OH, can be written in cloverleaf form. It exhibits many unorthodox features, perhaps the most strikking of which is the small size of the D-arm consisting of only 9 nucleotides. The anticodon loop contains 2 hypermodified nucleotides, U¹27 (probably 5-methoxycarbonylmethyluridine) and t⁶A30 (N-[N-(9-β-D-ribofuranosylpurin-6-yl)carbamoyl]threonine). The presence of U¹ in the first (“wobble”) position of the anticodon probably prevents the lysine tRNA from reading asparagine (AAY) codons. t⁶A, which is 3′-adjacent to the anticodon in most tRNAs recognizing codons starting with A, and other modified nucleosides occupy expected positions. We hypothesize that enzymes modifying the wobble position and the position 3′-adjacent to the anticodon recognize specific nucleotides in the anticodon.     

Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver.

The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequencing the bovine liver tRNA(Leu), the structure of the anticodon was determined as f5CmAA. In addition, the nucleotide sequence showed two primary structures differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs-Leu(f5CmAA) are the only tRNAs so far sequenced which contain f5Cm. The role of such a modified cytidine at the first position of the anticodon is discussed in terms of decoding properties for the UUG and UUA leucine codons. Recently, precise evidence was obtained for the presence of f5Cm at the same position in tRNAs(Leu)(NAA) isolated from rabbit and lamb liver. Therefore, the 2'-O-methyl-5-formyl modification of cytidine at position 34 could be a general feature of cytoplasmic tRNAs(Leu)(NAA) in mammals.     

Unusual anticodon loop structure found in E.coli lysine tRNA.

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.     

Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.     

Why termination tRNAs have not been found? Because they are hidden in the large ribosomal RNA

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such a recognition, making it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.     

Why Termination tRNAs Have Not Been Found? Because They Are Hidden in the Large Ribosomal RNA (A Hypothesis)

It is well known that protein synthesis in ribosomes on mRNA requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. The synthesis is assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by its anticodon is known. The termination factors are thought to do this. We discovered in the large ribosomal RNA two regions that, like tRNAs, contain the anticodon hairpin, but with triplets complementary to stop codons. By analogy, we called them termination tRNAs (Ter-tRNA1 and Ter-tRNA2), though they transport no amino acids, and suggested them to directly recognize stop codons. The termination factors only condition such recognition to make it specific and reliable (of course, they fulfill the hydrolysis of the ester bond between the polypeptide and tRNA). A strong argument in favor of our hypothesis came from vertebrate mitochondria. They acquired two new stop codons, AGA and AGG (in the standard code, they are two out of six arginine codons). We revealed that the corresponding anticodons appear in Ter-tRNA1.     

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.