Transfer RNA (abbreviatedtRNA and formerly referred to assRNA, forsoluble RNA[1]) is an adaptormolecule composed ofRNA, typically 76 to 90nucleotides in length (in eukaryotes).[2] In acell, it provides the physical link between thegenetic code inmessenger RNA (mRNA) and theamino acid sequence of proteins, carrying the correct sequence of amino acids to be combined by the protein-synthesizing machinery, theribosome. Each three-nucleotidecodon in mRNA iscomplemented by a three-nucleotideanticodon in tRNA. As such, tRNAs are a necessary component oftranslation, the biological synthesis of newproteins in accordance with the genetic code.
The process oftranslation starts with the information stored in the nucleotide sequence ofDNA. This is first transformed into mRNA, then tRNA specifies which three-nucleotide codon from the genetic code corresponds to which amino acid.[3] Each mRNA codon is recognized by a particular type of tRNA, which docks to it along a three-nucleotideanticodon, and together they form threecomplementarybase pairs.
On the other end of the tRNA is a covalent attachment to the amino acid corresponding to the anticodon sequence, with each type of tRNA attaching to a specific amino acid. Because the genetic code contains multiple codons that specify the same amino acid, there are several tRNA molecules bearing different anticodons which carry the same amino acid.
The covalent attachment to the tRNA3' end is catalysed by enzymes calledaminoacyl tRNA synthetases. During protein synthesis, tRNAs with attached amino acids are delivered to theribosome by proteins calledelongation factors, which aid in association of the tRNA with the ribosome, synthesis of the new polypeptide, and translocation (movement) of the ribosome along the mRNA. If the tRNA's anticodon matches the mRNA, another tRNA alreadybound to the ribosome transfers the growing polypeptide chain from its 3' end to the amino acid attached to the 3' end of the newly delivered tRNA, a reaction catalysed by the ribosome. A large number of the individual nucleotides in a tRNA molecule may bechemically modified, often bymethylation ordeamidation. These unusual bases sometimes affect the tRNA's interaction withribosomes and sometimes occur in theanticodon to alter base-pairing properties.[4]
Secondary cloverleaf structure of a tRNA encoding for phenylalanine.Tertiary structure of tRNA.CCA tail in yellow,acceptor stem in purple,variable loop in orange,D arm in red,anticodon arm in blue withanticodon in black,T arm in green.3D animated GIF showing the structure of phenylalanine-tRNA from yeast (PDB ID 1ehz). White lines indicate base pairing by hydrogen bonds. In the orientation shown, the acceptor stem is on top and the anticodon on the bottom.[5]
The structure of tRNA can be decomposed into itsprimary structure, itssecondary structure (usually visualized as thecloverleaf structure), and itstertiary structure[6] (all tRNAs have a similar L-shaped 3D structure that allows them to fit into theP andA sites of theribosome). The cloverleaf structure becomes the 3D L-shaped structure through coaxial stacking of the helices, which is a commonRNA tertiary structure motif. The lengths of each arm, as well as the loop 'diameter', in a tRNA molecule vary from species to species.[6][7]The tRNA structure consists of the following:
Theacceptor stem is a 7- to 9-base pair (bp) stem made by the base pairing of the 5′-terminal nucleotide with the 3′-terminal nucleotide (which contains the CCA tail used to attach the amino acid). The acceptor stem may contain non-Watson-Crick base pairs.[6][8]
TheCCA tail is acytosine-cytosine-adenine sequence at the 3′ end of the tRNA molecule. The amino acid loaded onto the tRNA byaminoacyl tRNA synthetases, to formaminoacyl-tRNA, is covalently bonded to the 3′-hydroxyl group on the CCA tail.[9] This sequence is important for the recognition of tRNA by enzymes and critical in translation.[10][11] In prokaryotes, the CCA sequence is transcribed in some tRNA sequences. In most prokaryotic tRNAs and eukaryotic tRNAs, the CCA sequence is added during processing and therefore does not appear in the tRNA gene.[12]
Theanticodon loop is a 5-bp stem whose loop contains theanticodon.[6]
TheTΨC loop is named so because of the characteristic presence of the unusual base Ψ in the loop, where Ψ ispseudouridine, a modifieduridine. The modified base is often found within the sequence 5'-TΨCGA-3', with the T (ribothymidine, m5U) and A forming a base pair.[13]
Thevariable loop orV loop sits between the anticodon loop and the ΨU loop and, as its name implies, varies in size from 3 to 21 bases. In some tRNAs, the "loop" is long enough to form a rigid stem, thevariable arm.[14] tRNAs with a V loop more than 10 bases long is classified as "class II" and the rest is called "class I".[15]
Ananticodon[16] is a unit of threenucleotides corresponding to the three bases of anmRNAcodon. Each tRNA has a distinct anticodon triplet sequence that can form 3complementarybase pairs to one or more codons for an amino acid. Some anticodons pair with more than one codon due towobble base pairing. Frequently, the first nucleotide of the anticodon is one not found on mRNA:inosine, which canhydrogen bond to more than one base in the corresponding codon position.[4]: 29.3.9 Ingenetic code, it is common for a single amino acid to be specified by all four third-position possibilities, or at least by bothpyrimidines andpurines; for example, the amino acidglycine is coded for by the codon sequences GGU, GGC, GGA, and GGG. Other modified nucleotides may also appear at the first anticodon position—sometimes known as the "wobble position"—resulting in subtle changes to the genetic code, as for example inmitochondria.[17] The possibility of wobble bases reduces the number of tRNA types required: instead of 61 types with one for each sense codon of the standard genetic code), only 31 tRNAs are required to translate, unambiguously, all 61 sense codons.[3][18]
A tRNA is commonly named by its intended amino acid (e.g.tRNA-Asn), by its anticodon sequence (e.g.tRNA(GUU)), or by both (e.g.tRNA-Asn(GUU) ortRNAAsn GUU).[19] These two features describe the main function of the tRNA, but do not actually cover the whole diversity of tRNA variation; as a result, numerical suffixes are added to differentiate.[20] tRNAs intended for the same amino acid are called "isotypes"; these with the same anticodon sequence are called "isoacceptors"; and these with both being the same but differing in other places are called "isodecoders".[21]
Aminoacylation is the process of adding an aminoacyl group to a compound. It covalently links anamino acid to the CCA 3′ end of a tRNA molecule.Each tRNA is aminoacylated (orcharged) with a specific amino acid by anaminoacyl tRNA synthetase. There is normally a single aminoacyl tRNA synthetase for each amino acid, despite the fact that there can be more than one tRNA, and more than one anticodon for an amino acid. Recognition of the appropriate tRNA by the synthetases is not mediated solely by the anticodon, and the acceptor stem often plays a prominent role.[22]Reaction:
Certain organisms can have one or more aminophosphate-tRNA synthetases missing. This leads to charging of the tRNA by a chemically related amino acid, and by use of an enzyme or enzymes, the tRNA is modified to be correctly charged. For example,Helicobacter pylori has glutaminyl tRNA synthetase missing. Thus, glutamate tRNA synthetase charges tRNA-glutamine(tRNA-Gln) withglutamate. An amidotransferase then converts the acid side chain of the glutamate to the amide, forming the correctly charged gln-tRNA-Gln.
The range of conformations adopted by tRNA as it transits the A/T through P/E sites on the ribosome. The Protein Data Bank (PDB) codes for the structural models used as end points of the animation are given. Both tRNAs are modeled as phenylalanine-specific tRNA fromEscherichia coli, with the A/T tRNA as a homology model of the deposited coordinates. Color coding as shown fortRNA tertiary structure. Adapted from.[23]
Theribosome has three binding sites for tRNA molecules that span the space between the tworibosomal subunits: theA (aminoacyl),[24]P (peptidyl), andE (exit) sites. In addition, the ribosome has two other sites for tRNA binding that are used duringmRNA decoding or during the initiation ofprotein synthesis. These are the T site (namedelongation factor Tu) and I site (initiation).[25][26] By convention, the tRNA binding sites are denoted with the site on thesmall ribosomal subunit listed first and the site on thelarge ribosomal subunit listed second. For example, the A site is often written A/A, the P site, P/P, and the E site, E/E.[25] The binding proteins like L27, L2, L14, L15, L16 at the A- and P- sites have been determined by affinity labeling by A. P. Czernilofsky et al. (Proc. Natl. Acad. Sci, USA, pp. 230–234, 1974).
Once translation initiation is complete, the first aminoacyl tRNA is located in the P/P site, ready for the elongation cycle described below. During translation elongation, tRNA first binds to the ribosome as part of a complex with elongation factor Tu (EF-Tu) or its eukaryotic (eEF-1) or archaeal counterpart. This initial tRNA binding site is called the A/T site. In the A/T site, the A-site half resides in thesmall ribosomal subunit where the mRNA decoding site is located. The mRNA decoding site is where themRNAcodon is read out during translation. The T-site half resides mainly on thelarge ribosomal subunit where EF-Tu or eEF-1 interacts with the ribosome. Once mRNA decoding is complete, the aminoacyl-tRNA is bound in the A/A site and is ready for the nextpeptide bond[27] to be formed to its attached amino acid. The peptidyl-tRNA, which transfers the growing polypeptide to the aminoacyl-tRNA bound in the A/A site, is bound in the P/P site. Once the peptide bond is formed, the tRNA in the P/P site is acylated, or has afree 3' end, and the tRNA in the A/A site dissociates the growing polypeptide chain. To allow for the next elongation cycle, the tRNAs then move through hybrid A/P and P/E binding sites, before completing the cycle and residing in the P/P and E/E sites. Once the A/A and P/P tRNAs have moved to the P/P and E/E sites, the mRNA has also moved over by onecodon and the A/T site is vacant, ready for the next round of mRNA decoding. The tRNA bound in the E/E site then leaves the ribosome.
The P/I site is actually the first to bind to aminoacyl tRNA, which is delivered by an initiation factor calledIF2 in bacteria.[26] However, the existence of the P/I site in eukaryotic or archaealribosomes has not yet been confirmed. The P-site protein L27 has been determined by affinity labeling by E. Collatz and A. P. Czernilofsky (FEBS Lett., Vol. 63, pp. 283–286, 1976).
Organisms vary in the number of tRNAgenes in theirgenome. For example, thenematode wormC. elegans, a commonly used model organism ingenetics studies, has 29,647 genes in itsnuclear genome,[28] of which 620 code for tRNA.[29][30] The budding yeastSaccharomyces cerevisiae has 275 tRNA genes in its genome. The number of tRNA genes per genome can vary widely, with bacterial species from groups such as Fusobacteria and Tenericutes having around 30 genes per genome while complex eukaryotic genomes such as the zebrafish (Danio rerio) can bear more than 10 thousand tRNA genes.[31]
In the human genome, which, according to January 2013 estimates, has about 20,848 protein coding genes[32] in total, there are 497 nuclear genes encoding cytoplasmic tRNA molecules, and 324 tRNA-derivedpseudogenes—tRNA genes thought to be no longer functional[33] (although pseudo tRNAs have been shown to be involved inantibiotic resistance in bacteria).[34] As with all eukaryotes, there are 22mitochondrial tRNA genes[35] in humans. Mutations in some of these genes have been associated with severe diseases like theMELAS syndrome. Regions in nuclearchromosomes, very similar in sequence to mitochondrial tRNA genes, have also been identified (tRNA-lookalikes).[36] These tRNA-lookalikes are also considered part of thenuclear mitochondrial DNA (genes transferred from the mitochondria to the nucleus).[36][37] The phenomenon of multiple nuclear copies of mitochondrial tRNA (tRNA-lookalikes) has been observed in many higher organisms from human to the opossum[38] suggesting the possibility that the lookalikes are functional.
Cytoplasmic tRNA genes can be grouped into 49 families according to their anticodon features. These genes are found on all chromosomes, except the 22 and Y chromosome. High clustering on 6p is observed (140 tRNA genes), as well as on chromosome 1.[33]
TheHGNC, in collaboration with the Genomic tRNA Database (GtRNAdb) and experts in the field, has approved unique names for human genes that encode tRNAs.
Typically, tRNAs genes from Bacteria are shorter (mean = 77.6 bp) than tRNAs from Archaea (mean = 83.1 bp) and eukaryotes (mean = 84.7 bp).[31] The mature tRNA follows an opposite pattern with tRNAs from Bacteria being usually longer (median = 77.6 nt) than tRNAs from Archaea (median = 76.8 nt), with eukaryotes exhibiting the shortest mature tRNAs (median = 74.5 nt).[31]
Genomic tRNA content is a differentiating feature of genomes among biological domains of life: Archaea present the simplest situation in terms of genomic tRNA content with a uniform number of gene copies, Bacteria have an intermediate situation and Eukarya present the most complex situation.[39] Eukarya present not only more tRNA gene content than the other two kingdoms but also a high variation ingene copy number among different isoacceptors, and this complexity seem to be due to duplications of tRNA genes and changes in anticodon specificity[citation needed].
Evolution of the tRNA gene copy number across different species has been linked to the appearance of specific tRNA modification enzymes (uridine methyltransferases in Bacteria, and adenosine deaminases in Eukarya), which increase the decoding capacity of a given tRNA.[39] As an example, tRNAAla encodes four different tRNA isoacceptors (AGC, UGC, GGC and CGC). In Eukarya, AGC isoacceptors are extremely enriched in gene copy number in comparison to the rest of isoacceptors, and this has been correlated with its A-to-I modification of its wobble base. This same trend has been shown for most amino acids of eukaryal species. Indeed, the effect of these two tRNA modifications is also seen incodon usage bias. Highly expressed genes seem to be enriched in codons that are exclusively using codons that will be decoded by these modified tRNAs, which suggests a possible role of these codons—and consequently of these tRNA modifications—in translation efficiency.[39]
Many species have lost specific tRNAs during evolution. For instance, both mammals and birds lack the same 14 out of the possible 64 tRNA genes, but other life forms contain these tRNAs.[40] For translating codons for which an exactly pairing tRNA is missing, organisms resort to a strategy calledwobbling, in which imperfectly matched tRNA/mRNA pairs still give rise to translation, although this strategy also increases the propensity for translation errors.[41] The reasons why tRNA genes have been lost during evolution remains under debate but may relate improving resistance to viral infection.[42] Because nucleotide triplets can present more combinations than there are amino acids and associated tRNAs, there is redundancy in the genetic code, and several different 3-nucleotide codons can express the same amino acid. This codon bias is what necessitates codon optimization.
The top half of tRNA (consisting of the T arm and the acceptor stem with 5′-terminal phosphate group and 3′-terminal CCA group) and the bottom half (consisting of the D arm and the anticodon arm) are independent units in structure as well as in function. The top half may have evolved first including the 3′-terminal genomic tag which originally may have marked tRNA-like molecules for replication in earlyRNA world. The bottom half may have evolved later as an expansion, e.g. as protein synthesis started in RNA world and turned it into a ribonucleoprotein world (RNP world). This proposed scenario is calledgenomic tag hypothesis. In fact, tRNA and tRNA-like aggregates have an important catalytic influence (i.e., asribozymes) on replication still today. These roles may be regarded as 'molecular (or chemical) fossils' of RNA world.[43] In March 2021, researchers reported evidence suggesting that an early form of transfer RNA could have been a replicatorribozyme molecule in the very early development of life, orabiogenesis.[44][45]
Evolution of type I and type II tRNAs is explained to the last nucleotide by the three 31 nucleotide minihelix tRNA evolution theorem, which also describes the pre-life to life transition on Earth.[46][47][48][49][50] Three 31 nucleotide minihelices of known sequence were ligated in pre-life to generate a 93 nucleotide tRNA precursor. In pre-life, a 31 nucleotide D loop minihelix (GCGGCGGUAGCCUAGCCUAGCCUACCGCCGC) was ligated to two 31 nucleotide anticodon loop minihelices (GCGGCGGCCGGGCU/???AACCCGGCCGCCGC; / indicates a U-turn conformation in the RNA backbone; ? indicates unknown base identity) to form the 93 nucleotide tRNA precursor. To generate type II tRNAs, a single internal 9 nucleotide deletion occurred within ligated acceptor stems (CCGCCGCGCGGCGG goes to GGCGG). To generate type I tRNAs, an additional, related 9 nucleotide deletion occurred within ligated acceptor stems within the variable loop region (CCGCCGCGCGGCGG goes to CCGCC). These two 9 nucleotide deletions are identical on complementary RNA strands. tRNAomes (all of the tRNAs of an organism) were generated by duplication and mutation.
Very clearly, life evolved from a polymer world that included RNA repeats and RNA inverted repeats (stem-loop-stems). Of particular importance were the 7 nucleotide U-turn loops (CU/???AA). After LUCA (the last universal common (cellular) ancestor), the T loop evolved to interact with the D loop at the tRNA “elbow” (T loop: UU/CAAAU, after LUCA). Polymer world progressed to minihelix world to tRNA world, which has endured for ~4 billion years. Analysis of tRNA sequences reveals a major successful pathway in evolution of life on Earth.
tRNA-derived fragments (or tRFs) are short molecules that emerge after cleavage of the mature tRNAs or the precursor transcript.[51][52][53][54] Both cytoplasmic and mitochondrial tRNAs can produce fragments.[55] There are at least four structural types of tRFs believed to originate from mature tRNAs, including the relatively long tRNA halves and short 5'-tRFs, 3'-tRFs and i-tRFs.[51][55][56] The precursor tRNA can be cleaved to produce molecules from the 5' leader or 3' trail sequences. Cleavage enzymes include Angiogenin, Dicer, RNase Z and RNase P.[51][52] Especially in the case of Angiogenin, the tRFs have a characteristically unusual cyclic phosphate at their 3' end and a hydroxyl group at the 5' end.[57] tRFs appear to play a role inRNA interference, specifically in the suppression of retroviruses and retrotransposons that use tRNA as a primer for replication. Half-tRNAs cleaved byangiogenin are also known as tiRNAs. The biogenesis of smaller fragments, including those that function aspiRNAs, are less understood.[58]
tRFs have multiple dependencies and roles; such as exhibiting significant changes between sexes, among races and disease status.[55][59][60] Functionally, they can be loaded on Ago and act through RNAi pathways,[53][56][61] participate in the formation of stress granules,[62] displace mRNAs from RNA-binding proteins[63] or inhibit translation.[64] At the system or the organismal level, the four types of tRFs have a diverse spectrum of activities. Functionally, tRFs are associated with viral infection,[65] cancer,[56] cell proliferation[57] and also with epigenetic transgenerational regulation of metabolism.[66]
tRFs are not restricted to humans and have been shown to exist in multiple organisms.[56][67][68][69]
Two online tools are available for those wishing to learn more about tRFs: the framework for the interactive exploration ofmitochondrial andnucleartRNA fragments (MINTbase)[70][71] and the relational database ofTransferRNA relatedFragments (tRFdb).[72] MINTbase also provides a naming scheme for the naming of tRFs calledtRF-license plates (or MINTcodes) that is genome independent; the scheme compresses an RNA sequence into a shorter string.
tRNAs with modified anticodons and/or acceptor stems can be used to modify the genetic code. Scientists have successfully repurposed codons (sense and stop) to accept amino acids (natural and novel), for both initiation (see:start codon) and elongation.
In 1990, tRNAfMet2 CUA (modified from the tRNAfMet2 CAU genemetY) was inserted intoE. coli, causing it to initiate protein synthesis at the UAG stop codon, as long as it is preceded by a strongShine-Dalgarno sequence. At initiation it not only inserts the traditionalformylmethionine, but also formylglutamine, as glutamyl-tRNA synthase also recognizes the new tRNA.[73] The experiment was repeated in 1993, now with an elongator tRNA modified to be recognized by themethionyl-tRNA formyltransferase.[74] A similar result was obtained inMycobacterium.[75] Later experiments showed that the new tRNA was orthogonal to the regular AUG start codon showing no detectable off-target translation initiation events in a genomically recodedE. coli strain.[76]
Ineukaryotic cells, tRNAs aretranscribed byRNA polymerase III as pre-tRNAs in the nucleus.[77]RNA polymerase III recognizes two highly conserved downstream promoter sequences: the 5′ intragenic control region (5′-ICR, D-control region, or A box), and the 3′-ICR (T-control region or B box) inside tRNA genes.[2][78][79]The first promoter begins at +8 of mature tRNAs and the second promoter is located 30–60 nucleotides downstream of the first promoter. The transcription terminates after a stretch of four or morethymidines.[2][79]
Bulge-helix-bulge motif of a tRNA intron
Pre-tRNAs undergo extensive modifications inside the nucleus. Some pre-tRNAs containintrons that are spliced, or cut, to form the functional tRNA molecule;[80] in bacteria these self-splice, whereas in eukaryotes andarchaea they are removed by tRNA-splicingendonucleases.[81] Eukaryotic pre-tRNA contains bulge-helix-bulge (BHB) structure motif that is important for recognition and precise splicing of tRNA intron by endonucleases.[82] This motif position and structure are evolutionarily conserved. However, some organisms, such as unicellular algae have a non-canonical position of BHB-motif as well as 5′- and 3′-ends of the spliced intron sequence.[82]The 5′ sequence is removed byRNase P,[83] whereas the 3′ end is removed by thetRNase Z enzyme.[84]A notable exception is in thearchaeonNanoarchaeum equitans, which does not possess an RNase P enzyme and has a promoter placed such that transcription starts at the 5′ end of the mature tRNA.[85]The non-templated 3′ CCA tail is added by anucleotidyl transferase.[86]Before tRNAs areexported into thecytoplasm by Los1/Xpo-t,[87][88] tRNAs areaminoacylated.[89]The order of the processing events is not conserved.For example, inyeast, the splicing is not carried out in the nucleus but at the cytoplasmic side ofmitochondrial membranes.[90]
Interference with aminoacylation may be useful as an approach to treating some diseases: cancerous cells may be relatively vulnerable to disturbed aminoacylation compared to healthy cells. The protein synthesis associated with cancer and viral biology is often very dependent on specific tRNA molecules. For instance, for liver cancer charging tRNA-Lys-CUU with lysine sustains liver cancer cell growth and metastasis, whereas healthy cells have a much lower dependence on this tRNA to support cellular physiology.[100] Similarly, hepatitis E virus requires a tRNA landscape that substantially differs from that associated with uninfected cells.[101] Hence, inhibition of aminoacylation of specific tRNA species is considered a promising novel avenue for the rational treatment of a plethora of diseases.
^Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J. (2004).Molecular Cell Biology. WH Freeman: New York. 5th ed.ISBN978-0716743668[page needed]
^Ramos A, Barbena E, Mateiu L, del Mar González M, Mairal Q, Lima M, Montiel R, Aluja MP, Santos C, et al. (November 2011). "Nuclear insertions of mitochondrial origin: Database updating and usefulness in cancer studies".Mitochondrion.11 (6):946–953.doi:10.1016/j.mito.2011.08.009.PMID21907832.
^White RJ (March 1997). "Regulation of RNA polymerases I and III by the retinoblastoma protein: a mechanism for growth control?".Trends in Biochemical Sciences.22 (3):77–80.doi:10.1016/S0968-0004(96)10067-0.PMID9066256.
