The concept of RNA-assisted protein folding: the role of tRNA
© Biro; licensee BioMed Central Ltd. 2012
Received: 11 March 2012
Accepted: 2 April 2012
Published: 2 April 2012
We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding.
The concept of nucleic acid-assisted protein folding (nucleic acid chaperons) was first published in 2005 , when it was suggested that mRNAs participate in the formation of the tertiary structure of their coded proteins; this function is in addition to the well-recognized determination of the amino acid sequence of the proteins, as described in Nirenberg's Genetic Code. Messenger RNAs often have a tertiary structure that provides spatial information for protein folding, i.e. they are nucleic acid chaperons . Nucleic acid-assisted protein folding requires that mRNA and coded peptides remain in contact with each other, even after the polymerization of the amino acids during translation. The original model described RNA-assisted protein folding, but the role of tRNA was not properly elucidated.
The role of the t-RNAs is to "translate" between mRNA and the encoded peptide, which means that tRNAs recognizes the codons in mRNA and arrange the amino acids in the order of the codons for polymerization by the protein synthetase. This function could be performed by a much smaller oligonucleotide containing only four nucleotides, three for the anticodon and one for binding the corresponding amino acid. However tRNAs are much larger, about 76 nucleotides in length.
The abundance of tRNA is another mystery. The 64 different codons require 64 different "mediators", but there are many more. There are, for example, 359 different cytoplasmic tRNAs in human cells, and many more when genomic variants are included. The generous size and lavish redundancy suggest that the function of tRNAs is far more complex than the passive connecting of codons with their amino acids that has been recognized to date.
The aim of this work is to identify additional functions for tRNAs in translation, and to explain their involvement and role in nucleic acid-mediated protein folding.
Results and discussion
The existence of tRNA was hypothesized by Francis Crick in 1956 [3, 4] to explain the transfer of information (translation) from nucleic acids to proteins, which are two structurally very different molecules. The first tRNA sequences were described and structures were suggested in 1965 by Holley et al. . Transfer RNA was required to "fill the gap" between amino acids and codons, or spatially "convert" the large codons (3 nucleotides, ~1 nm in length) to amino acids (single molecules, about 1/3 nm length when incorporated in proteins).
The structural complexity of tRNA is reminiscent of that of a protein, with 71 out of 76 bases participating in a stacking interaction (42 of the bases have a double helical stem structure). A series of specific 9 bp sequences crosslink the tertiary structure of the tRNA by interacting with bases from a different stem and loop region of the molecule. All of these 9-bp interactions are non-Watson-Crick associations and are highly conserved, which makes it likely that all tRNA molecules have similar structures, although only a few have been crystallized and their structures determined.
To provide a one-to-one correspondence between tRNA molecules and the codons that specify amino acids, 61 types of tRNA molecules would be required per cell. However, many cells contain fewer than the predicted 61 types of tRNAs because the wobble base is capable of binding to several, though not necessarily all, of the codons that specify a particular amino acid. A minimum of 31 tRNAs are required to translate, unambiguously, all 61 sense codons of the standard genetic code [6, 7].
However, the total number of tRNAs is much larger than the number of codons. In the human genome, which according to estimates has about 27,161 genes , there are in total about 4,421 non-coding RNA genes, which include tRNA genes. There are 22 mitochondrial tRNA genes, 497 nuclear genes encoding cytoplasmic tRNA molecules, and 324 tRNA-derived putative pseudogenes .
This means that there are, on average, eight slightly different cytoplasmic tRNAs for every possible codon. Therefore, the size and the redundancy of tRNAs are very large for the "tiny" function that is accepted today. Identifying new functions and explaining the size and redundancy of tRNA is one of the goals of our study.
The tRNA database that we used [10, 11] lists 359 human tRNA sequences. There are tRNAs corresponding not only to the 20 amino acids, but also to the initiation (Ini, CAT, Met) and termination (Sec, TCA) sites. Many codons have no tRNA representations in this database, including AAA (Lys), ACA (Thr), ACC (Thr), ACT (Thr), ATC (Ile), ATG (Met), CTA (Leu), GAC (Asp), GAG (Glu), GCG (Ala), GGA (Gly), GGC (Gly), GGG (Gly), GGT (Gly), TTA (Leu). Each of the remaining 49 codons is represented by an average of 7-8 different tRNAs.
(The relatively large numbers of tRNAs may be readily explained by the fact, that many fundamentally important genes code isoforms of the same proteins (for example the enzymes of glycolysis), so that a mutation which result a loss of function will not be immediately lethal. In case of only one tRNA alternative, this would have been way too risky for life.)
It is important to keep in mind that almost all of these tRNAs have been identified using bioinformatics tools that were looking for sequence characteristics assigned to real tRNAs. However, their existence and biological functions have yet to be confirmed by biochemists. Even the Protein and Nucleic Acid Databases (PDB, NDB, [12, 13]) are surprisingly empty of real tRNA structures, although many tRNA-associated proteins have been the subject of structural studies.
When speaking about tRNA the reader has to keep in mind that translation is still a very "hot" subject for many scientists, "the dark side of molecular biology"  driven by a powerful paradigm. This 50 year old paradigm suggests that translation is a "tape reading" where there is a "tape" (mRNA) and step-by-step reading it (tRNA) provides proteins on the surface of ribosomes. The ribosome has a door "in" (A site) and a door "out" (P site) [15, 16]. This "cartoon guide to translation"  very strictly separates the "RNA World" from the "Protein World", where the only connection permitted is the tRNA (the "adaptor"). However the connection between codons and amino acids (provided by tRNA) is not logical or the result of evolution, but accidental, a so called "frozen accident"  and codons and amino acids never interact with each other. Consequently, in every published structure, the distance between the amino acid and the anticodon is well over 50 Å
Although this mechanical model is supported by structural studies [18–24] the reality is that crystal structures present only snapshots of thermodynamically stable conformations. Solution and computational methods provide evidence for the inherent flexibility of tRNA structure under a variety of conditions and for differing tRNA species. Transfer RNAs perform a wide range of motion and conformational changes and allosteric transition during the process of translation. [25–27] Lapointe, Alexander, Caulfield]. Molecular Dynamic methods, like simulations of Cryo-EM Microscopy and X-Ray Data to explore intermediate conformational space, often provide previously unidentified structures which are very different from the canonical cloverleaf-like manifestations. [27–30]. Interaction with other macromolecules is known to cause structural alteration in tRNAs. [31–33]. The tRNA structure is sensitive for changes in the molecular environment, post-transcriptional modifications. A single base mutation is sufficient to change the "cloverleaf" configuration into a "hairpin"-like folding [29, 34].
An estimation of the folding energy (Quickfold program ) indicates that all human tRNAs have negative folding energies (-26.1 ± 4.42 kcal/mole, SD, n = 359). However, even randomized tRNA sequences possess significant folding energies (-21.1 ± 4.79 kcal/mole, SD, n = 369). The difference between these two values (-4.95 ± 5.66 kcal/mole, SD, n = 369), the Free Folding Energy, FFE, is rather small, only approximately 19% of the total folding energy, and there are even numerous positive values. This indicates that the majority of the total folding energy of tRNAs is provided by the nucleotide composition, and not by the nucleotide sequence itself, i.e. tRNAs may fold into numerous different structures in addition to the typical cloverleaf configuration.
The common feature of all tRNAs is that their primary structures contain a large number of complementary residues and these Watson-Crick-type interactions, within the same sequence, provide a characteristic tertiary structure. This property suggests that complementary interactions do exist not only within but also between tRNAs, as they have a high degree of similarity. The question of interactions between tRNAs was studied here using in silico and hybridization (melting) studies.
Interaction between tRNAs is thermodynamically favored. Not unexpectedly, the tRNA hybridizes with itself (dG values for intact and randomized sequences are -43.7 ± 8.22 kcal/mole, SD, n = 359 and -33.5 ± 6.76, SD, n = 359, respectively, and the difference, the Free Hybridization Energy, FHE, is -10.2 ± 7.18 kcal/mole, SD, n = 359). The codon- and amino acid-related distribution of dG values for hybridization is similar to the distribution of dG values for folding in that there is a wide range, and even some positive values; however, the correlation between FFE and FHE is not significant (data not shown). The mean dG of hybridization is much higher than the mean dG of folding, indicating that the potential for tRNA interactions (Watson-Crick pairs) is not fully utilized during folding compared to hybridization.
The idea that tRNAs interact with each other during translation is not new. It was first suggested by Carl Woese in 1970  in his "reciprocating ratchet mechanism" for protein synthesis which depends upon conformational changes in tRNA and allosteric transitions in place of translocation.
Model of tRNA structures and interactions
The native, free, cytoplasmic tRNA has a cloverleaf structure, dG:~-26 kcal/mole.
The tRNAs come into intimate contact with each other in the mRNA-rRNA-tRNA complex. This proximity destabilizes the cloverleaf structure and transforms the molecules into thermodynamically more favored homo- and hetero-dimers (dG: ~-43.7 and ~-32.8 kcal/mole, respectively. p < 0.001). Statistical analyses were performed using Student's t-test .
However, the dimer is not stable either, because the arrival of a 3rd tRNA, which has affinity for the 2nd tRNA, provides competition with the 1st tRNA and destabilizes the bonds between the 1st and 2nd tRNAs in the first dimer.
The 1st tRNA is released from the first dimer and a 2nd dimer is formed by the 2nd and 3rd tRNAs.
The free 1st tRNA refolds into 5'-tail (dG:~-10.7 kcal/mole), 3'-tail (dG: ~-10 kcal/mole), double tail (5'- and 3' tails together dG: ~-20.7 kcal/mole), and hairpin (dG:~-20.6 kcal/mole) forms, and finally to the original cloverleaf structure (dG: ~-26.1 kcal/mole); the structural cycle is then completed.
The cycle repeats itself with the 2nd, 3rd ... etc. tRNAs as the translation process continues to its completion.
Dimerization or aggregation of naturally-occurring tRNAs in vitro is a known phenomenon, but typically occurs under non-physiological conditions [46–49]. Unmodified tRNA transcripts, as well as mature tRNAs, can undergo complex formation . The dimeric tRNA forms are often associated with pathological conditions . However, we suggest that dimerization of tRNA is a normal, natural phenomenon.
The possibility of specific, high affinity interactions between codons and encoded amino acids has been the subject of long and intense debate, with a great many personalities involved. Francis Crick vehemently rejected this connection and stated that any connection between codons and encoded amino acids is only the result of a "frozen accident" . His view, owing to his strong personality and his Nobel Prize in 1962, has been and remains very successful even, and affects the perceptions of the scientific community on this question. On the other hand, there is very strong evidence to suggest that amino acids are evolutionarily strongly connected to their codons [7, 51–53], and they preferentially collocate with each other in protein-nucleic acid complexes . We find that specific codon-amino acid interactions are necessary, at least for some "dedicated" examples that mark structurally critical points in the peptide and mRNA sequences and perform the correct protein folding under the guidance of a nucleic acid. The literature , as well as our studies , indicates that Arg, Lys, Asn and Gln are examples of these "dedicated" amino acids.
We suggest that tRNAs have a far more complex role in translation than functioning only as passive adaptors. They are able to change form and interact with each other, and this change seems to be necessary for mRNA-assisted protein folding to be carried out. These new roles explain the large size and generous redundancy of tRNAs.
- Biro JC: Nucleic acid chaperons: a theory of an RNA-assisted protein folding. Theor Biol Med Model. 2005, 2: 35-10.1186/1742-4682-2-35.PubMed CentralView ArticlePubMedGoogle Scholar
- Biro JC: The Proteomic Code: a molecular recognition code for proteins. Theor Biol Med Model. 2007, 4: 45-10.1186/1742-4682-4-45.PubMed CentralView ArticlePubMedGoogle Scholar
- Crick FHC: On Degenerate Templates and the Adaptor Hypothesis: A Note for the RNA Tie Club. [http://profiles.nlm.nih.gov/SC/B/B/G/F/_/scbbgf.pdf], 1956 - last accessed 2013 01 12
- Rajbhandary UL, Köhrer C: Early days of tRNA research: discovery, function, purification and sequence analysis. J Biosci. 2006, 31: 439-451. 10.1007/BF02705183.View ArticlePubMedGoogle Scholar
- Holley RW, Apgar J, Everett GA, Madison JT, Marquisee M, Merrill SH, Penswick JR, Zamir A: Structure of a ribonucleic acid. Science. 1965, 147: 1462-1465. 10.1126/science.147.3664.1462.View ArticlePubMedGoogle Scholar
- Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J: Molecular Biology of the Cell. 2004, New York: WH Freeman, 5Google Scholar
- Crick FHC: The origin of the genetic code. J Mol Biol. 1968, 38: 367-379. 10.1016/0022-2836(68)90392-6.View ArticlePubMedGoogle Scholar
- Ensembl release 48 - Dec 2007. [http://www.ensembl.org]
- Lander E: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.View ArticlePubMedGoogle Scholar
- tRNAdb 2009. last accessed 2012 01 12, [http://trnadb.bioinf.uni-leipzig.de/Welcome]
- Jühling F, Mörl M, Hartmann RK, Sprinzl M, Stadler PF, Pütz J: tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res. 2009, D159-D162. 37 DatabaseGoogle Scholar
- Protein Data Bank. Last accessed 2012 01 12, [http://www.rcsb.org/pdb/home/home.do]
- Nucleic Acid Database. Last accessed 2012 01 12, [http://ndbserver.rutgers.edu/]
- Woese CR: Translation: in retrospect and prospect. RNA. 2001, 7: 1055-1067. 10.1017/S1355838201010615.PubMed CentralView ArticlePubMedGoogle Scholar
- Watson JD: The synthesis of proteins upon ribosomes. Bull Soc Chim Biol. 1964, 46: 1399-1425.PubMedGoogle Scholar
- Watson JD: Molecular biology of the gene. 1976, Menlo Park, California: WA Benjamin Inc.Google Scholar
- Crick F: Life Itself. Its Origin and Nature. 1981, Simon & Schuster, New YorkGoogle Scholar
- VanLoock MS, Easterwood TR, Harvey SC: Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site. J Mol Biol. 1999, 285: 2069-2078. 10.1006/jmbi.1998.2442.View ArticlePubMedGoogle Scholar
- Easterwood TR, Harvey SC: Modeling the structure of the ribosome. Biochem Cell Biol. 1995, 73: 751-756. 10.1139/o95-083.View ArticlePubMedGoogle Scholar
- Frank J: Three-dimensional electron microscopy of macromolecular assemblies: visualization of biological molecules in their native state. (Google eBook). 2006, Oxford: Oxford University PressGoogle Scholar
- Fu J, Munro JB, Blanchard SC, Frank J: Cryo-EM structures of the ribosome complex in intermediate states during tRNA translocation. Proc Natl Acad Sci USA. 2011, 108: 4817-4821. 10.1073/pnas.1101503108.PubMed CentralView ArticlePubMedGoogle Scholar
- Yonath AE: Hibernating Bears, Antibiotics and the Evolving Ribosome. Nobel Lecture, Nobelprize.org. 2012Google Scholar
- Ramakrishnan V: Unraveling the Structure of the Ribosome. Nobel Lecture, Nobelprize.org. 2012Google Scholar
- Steitz TA: From the Structure and Function of The Ribosome to New Antibiotics. Nobel Lecture, Nobelprize.org. 2012Google Scholar
- Lapointe J, Brakier-Gingras L: Translation mechanisms. 2003, Georgetown, TX USA: Kluwer Academic/Plenum PublishersGoogle Scholar
- Alexander RW, Eargle J, Luthey-Schulten Z: Experimental and computational determination of tRNA dynamics. FEBS Lett. 2010, 584: 376-386. 10.1016/j.febslet.2009.11.061.View ArticlePubMedGoogle Scholar
- Caulfield TR, Devkota B, Rollins GC: Examinations of tRNA Range of Motion Using Simulations of Cryo-EM Microscopy and X-Ray Data. Journal of Biophysics. 2011, Article ID 219515, 11 pagesGoogle Scholar
- Fu J, Munro JB, Blanchard SC, Frankac J: Cryoelectron microscopy structures of the ribosome complex in intermediate states during tRNA translocation. Proc Natl Acad Sci USA. 2011, 108: 4817-4821. 10.1073/pnas.1101503108.PubMed CentralView ArticlePubMedGoogle Scholar
- Giegé R, Jühling F, Pütz J, Stadler P, Sauter C, Florentz C: Structure of transfer RNAs: similarity and variability. WIREs RNA. 2012, 3: 37-61. 10.1002/wrna.103.View ArticlePubMedGoogle Scholar
- Jonikas MA, Radmer RJ, Laederach A, Das R, Pearlman S, Herschlag D, Altman RB: Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters. RNA. 2009, 15: 189-199. 10.1261/rna.1270809.PubMed CentralView ArticlePubMedGoogle Scholar
- Rees B, Cavarelli J, Moras D: Conformational flexibility of tRNA: structural changes in yeast tRNAAsp upon binding to aspartyl-tRNA synthetase. Biochimie. 1996, 78: 624-631. 10.1016/S0300-9084(96)80008-3.View ArticlePubMedGoogle Scholar
- Sissler M, Helm M, Frugier M, Giege R, Florentz C: Aminoacylation properties of pathology-related human mitochondrial tRNALys variants. RNA. 2004, 10: 841-853. 10.1261/rna.5267604.PubMed CentralView ArticlePubMedGoogle Scholar
- Patkowski P, Gulari E, Chu B: Long range tRNA-tRNA electrostatic interactions in salt-free and low-salt tRNA solutions. J Chem Phys. 1980, 73: 4178-4185. 10.1063/1.440725.View ArticleGoogle Scholar
- Helm M, Giegé R, Florentz C: A Watson-Crick base-pair disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. Biochemistry. 1999, 38: 13338-13346. 10.1021/bi991061g.View ArticlePubMedGoogle Scholar
- MFold Server. last accessed 2012 01 12, [http://mfold.rna.albany.edu/?q=mfold]
- Woese C: Molecular mechanics of translation: a reciprocating ratchet mechanism. Nature. 1970, 226: 817-820. 10.1038/226817a0.View ArticlePubMedGoogle Scholar
- El'skaya A, Negrutskii B: The interaction between biologically inactive tRNA conformers and leucyl-tRNA synthetase from rabbit liver. Eur J Biochem. 1987, 164: 65-69. 10.1111/j.1432-1033.1987.tb10993.x.View ArticlePubMedGoogle Scholar
- Kobitski AY, Hengesbac M, Helm M, Nienhaus GU: Sculpting of an RNA Conformational Energy Landscape by a Methyl Group Modification - A Single-Molecule FRET Study. Angew Chem Int. 2008, 47: 4326-4330. 10.1002/anie.200705675.View ArticleGoogle Scholar
- Madore E, Florentz C, Giege R, Lapointe J: Magnesium-dependent alternative foldings of active and inactive Escherichia coli tRNAGlu revealed by chemical probing. Nucleic Acid Research. 1999, 27: 3583-3688. 10.1093/nar/27.17.3583.View ArticleGoogle Scholar
- Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM: Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell. 2006, 21: 87-96. 10.1016/j.molcel.2005.10.036.View ArticlePubMedGoogle Scholar
- Engelke DR, Hopper AK: Modified View of tRNA: Stability amid Sequence Diversity. Mol Cell. 2006, 21: 87-96. 10.1016/j.molcel.2005.10.036. comments on Mol CellView ArticleGoogle Scholar
- Jorgensen T, Siboska GE, Wikman FP, Clark BFC: Different conformations of tRNA in the ribosomal P-site and A-site. Eur J Biochcm. 1985, 153: 203-209. 10.1111/j.1432-1033.1985.tb09287.x.View ArticleGoogle Scholar
- Miyazawa T, Yokoyama S: Conformational aspects and functions of tRNA. Proc Int Symp Biomol Struct Interactions Suppl J Biosci. 1985, 8: 731-737.Google Scholar
- Kholod NS: Dimer formation by tRNAs. Biochemistry (Mosc). 1999, 64: 298-306.Google Scholar
- Student's t-Tests. last accessed 2012 01 12, [http://www.physics.csbsju.edu/stats/t-test.html]
- Loehr JS, Keller EB: Dimers of alanine transfer RNA with acceptor activity. Proc Natl Acad Sci USA. 1968, 1968 (61): 1115-1122.View ArticleGoogle Scholar
- Yang SK, Söll DG, Crothers DM: Properties of a dimer of tRNATyr. Biochemistry. 1972, 11: 2311-2320. 10.1021/bi00762a016.View ArticlePubMedGoogle Scholar
- Kholod N: Dimer formation by tRNAs. Biochem Mosc. 1999, 64: 298-306.Google Scholar
- Madore E, Florentz C, Giege R, Lapointe J: Magnesium-dependent alternative foldings of active and inactive Escherichia coli tRNA(Glu) revealed by chemical probing. Nucleic Acids Res. 1999, 27: 3583-3588. 10.1093/nar/27.17.3583.PubMed CentralView ArticlePubMedGoogle Scholar
- Roy MD, Wittenhagen LM, Kelley SO: Structural probing of a pathogenic tRNA dimer. RNA. 2005, 11: 254-260. 10.1261/rna.7143305.PubMed CentralView ArticlePubMedGoogle Scholar
- Biro JC: Principia Bi®o-Informatica. The Proteomic Code. 2009, Homulus Foundation, Free download from [http://www.janbiro.com] - last accessed 2012 01 12Google Scholar
- Woese CR: The Genetic Code: The Molecular Basis for Gene Expression. 1967, New York: Harper & Row, Chapters 6-7: 156-160.Google Scholar
- Biro JC, Benyó B, Sansom C, Szlávecz A, Fördös G, Micsik T, Benyó Z: A common periodic table of codons and amino acids. Biochem Biophys Res Commun. 2003, 306: 408-415. 10.1016/S0006-291X(03)00974-4.View ArticlePubMedGoogle Scholar
- Biro JC, Biro JMK: Frequent occurrence of recognition site-like sequences in the restriction endonucleases. BMC Bioinforma. 2004, 5: 30-10.1186/1471-2105-5-30.View ArticleGoogle Scholar
- Sathyapriya R, Vishveshwara S: Interaction of DNA with clusters of amino acids in proteins. Nucl Acids Res. 2004, 32: 4109-4118. 10.1093/nar/gkh733.PubMed CentralView ArticlePubMedGoogle Scholar
- Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31: 3406-3415. 10.1093/nar/gkg595.PubMed CentralView ArticlePubMedGoogle Scholar
- Markham NR, Zuker M: UNAFold: Software for Nucleic Acid Folding and Hybridization. Data, Sequence Analysis, and Evolution. Bioinformatics: Volume 2. Edited by: Keith J. 2008, Humana Press Inc. Totowa, NJ, USA, Chapter 1: 3-31.Google Scholar
- Markham NR, Zuker M: DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res. 2005, 33: W577-W581. 10.1093/nar/gki591.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.