tRNA: Structure, Biosynthesis, and Function

Biochemical studies of protein synthesis in vitro got under way in the early 1950s. The fundamental concept arose that the specificity in protein synthesis was primarily governed by the loading of every amino acid onto a "cognate" soluble RNA by an enzyme specific for that amino acid. The RNAs involved began to be known as transfer RNAs (tRNAs), and the activating enzymes came to be known as aminoacyl-tRNA synthetases. With the elucidation of the genetic code in DNA, "the code in tRNA" emerged as a central problem in the molecular biology of protein biosynthesis, and structure and function in tRNA became the focus of attention in many laboratories. In the tRNA field, Abelson and Miller and their colleagues have carried out studies on aminoacylation specificity of tRNAs in vivo, using synthetic genes for suppressor tRNAs. This approach, together with RNA synthesis, for investigation of the sequence-dependent aminoacylation of RNA oligonucleotides in vitro has proved most useful for studying aminoacyl tRNA synthetases-tRNA recognitions in vivo. Totally synthetic genes lend themselves well to systematic mutagenesis by the principle of fragment (cassette) replacement. Structure-function studies on integral membrane proteins such as bacteriorhodopsin, sensory rhodopsin, and the vertebrate photoreceptor rhodopsin have used exclusively the synthetic gene approach. A large number of chimeric genes of visual color pigments were synthesized by Oprian and colleagues for precise studies of spectral tuning in vision.

This chapter reviews the basis for the current view of eukaryotic tRNA promoters and transcription machinery, and discusses studies of mechanistic and regulatory strategies. Recent insights into the nature of the polymerase III transcription machinery give substance to these speculations and suggest a specific role for 5' flanking promoter elements. The discussion of the polymerase III transcription machinery first focuses on the two traditional factor fractions TFIIIB and TFIIIC and then on individual components recently resolved from these fractions. In the discussion, standard TFIIIB and TFIIIC nomenclature are used, but, keeping the heterogeneity of these fractions in mind, “F” are taken to mean “fraction” rather than “factor.” Also, it may be useful to avoid assumptions and consider the transcriptionally active polypeptides in these fractions as independent transcription factors, rather than as tightly associated subunits. Given the number of macromolecules involved in class III transcription, genetic approaches are likely to be essential for identifying all of the players. An intriguing focus of current work is the regulation of tRNA transcription. There are now a number of examples of enhanced production of particular tRNAs in response to cellular differentiation and growth conditions.

This chapter is primarily a progress report on work with the enzyme from Escherichia coli. RNase P, the endonuclease responsible for the biosynthesis of the 5' termini of mature tRNA, is a ribonucleoprotein. The chapter primarily focuses on relationships between the structure and function of the subunits of RNase P, as determined from studies with the enzyme from eubacterial sources. Two important goals of current research on RNase P are identification of the active site of the enzyme and elucidation of the details of the chemical reaction governed by it. Several lines of evidence have implicated the region in M1 RNA that contains at least nucleotides 60 to 92, 230 to 260, and 290 to 360 as being essential for catalysis. The evidence comes from analysis of deletion mutants, studies of the binding of both individual tRNA precursors and the protein cofactor to M1 RNA, and studies of the binding of divalent metal ions to M1 RNA. Some point mutations that significantly affect the activity of M1 RNA are also located in these regions. A summary of these results is shown in the chapter. The protein cofactor of RNase P from E. coli (C5 protein) is a highly basic molecule of 119 amino acids, with a molecular mass of 13,800 daltons.

This chapter talks about primary, secondary, and tertiary structures of tRNAs. What is striking about the tRNA molecule is its extreme variability in primary and secondary structures. Every single invariant or semi-invariant position has numerous exceptions depending on the origin of the cell from which the tRNA is extracted. Several elements of the classical cloverleaf structure can altogether disappear, evidently as long as the amino acid and anticodon parts are maintained. Thus, the variable region, the D-arm, and the T-arm can be missing or severely amputated without apparent disfunctionality of the tRNA. This diversity at the sequence and 2D-structure levels must clearly manifest fest itself at the 3D-structure level also. This is discernible in some crystal structures and, especially, in recently modelled structures, although in the latter case one cannot reach the same degree of confidence. Nature has tinkered around every tertiary interaction responsible for maintaining the famous L-structure of tRNAs. The tRNA-like structures found at the 3' end of viral RNAs display some of the most extreme cases of divergence from canonical tRNA structure.

This chapter summarizes the structural and functional mimicry of tRNA by plant viral tRNA-like structures, and emphasizes how studies on these structures were innovative in the domain of RNA folding and beneficial to the better understanding of canonical tRNA aminoacylation. It also discusses current ideas about the biological significance of these aminoacylatable structures as well as other viral RNA sequences mimicking tRNA, during the life cycles of their carrier viruses.

This chapter focuses on the mechanisms by which the post-transcriptional modifications regulate the codon recognition of tRNAs, primarily from the viewpoint of conformational characteristics of modified nucleosides. Post-transcriptional modifications are heavily involved in the specificities of codon recognition and also of aminoacylation. Interestingly, the roles of the modifications in the anticodon are in many cases the altered conformational properties such as conformational "rigidity" and "flexibility," which directly result in the rigidity or flexibility in codon recognition, although the chemical structures of the modified nucleosides are so much different from each other. It is quite natural because the "wobble" of the base from the original location for the Watson-Crick base pair is essential for non-Watson-Crick base pairing. In the future, more direct structural studies should be done on the anticodon-codon recognition in the decoding center of the ribosome. In addition to such studies at the level of molecular structures, biological studies on the roles of posttranscriptional modifications are required. For example, Q is mostly conserved from bacteria to higher eukaryotes but is missing in tRNAs from Mycoplasma and mitochondria. If the Q modification is not indispensable for protein synthesis, it is a wonder that why many organisms have to have such a complicated hypermodification. The real biological role of Q may be to play an essential role in other unknown functions of tRNA. For answering this question, more biological approaches such as gene targeting of modification enzymes in mammalian systems appear to be important and are therefore in progress in laboratories.

Translation of the genetic message into proteins implies the precise correspondence between the 64 base triplets and the 20 canonical amino acids. In this process, the tRNAs play a central role by providing the nascent polypeptide with the amino acids by which they are esterified, in response to codons on the mRNA. The pairing of mRNA codon to tRNA anticodon is independent of the nature of the amino acid esterified to the tRNA. Therefore, the accuracy of the tRNA aminoacylation reaction, ensured by the aminoacyl-tRNA synthetases (aaRS), is of first importance in all living cells, since it will govern, to a large extent, the fidelity of the translation process. Much work has been done to understand how aaRS achieve high accuracy of tRNA aminoacylation while maintaining a sufficiently high rate of catalysis, generally in the order of several turnovers per second.. Since the early description of aaRS, most of the studies have focused on the kinetic mechanisms of action of the aaRS. More recently, with the availability of the three-dimensional structure of several tRNAs and synthetases and the possibility of generating variants of these macromolecules, a static picture of their specific interaction at the atomic level has emerged. Two main functions are carried out by an aaRS: the activation of the amino acid and the recognition of the tRNA molecule. In addition, association between protomers must be ensured. The present knowledge indicates that each of these functions is distributed along the aaRS polypeptide through the formation of specialized domains.

This chapter describes the current understanding of the structural features in tRNA that determine the specificity of the interaction with the aminoacyl-tRNA synthetase (aaRS), and outlines future research in this area. Early methods of sequence comparison to predict tRNA identity determinants relied only on the structural similarities among isoacceptor tRNAs discerned by visual inspection of the sequences. Positions where the same nucleotide occurs in all isoacceptor species were considered more predictive of tRNA identity than were positions where the nucleotide differed among the isoacceptors. The newer approach relies on computer analysis of tRNA sequences and identifies not only conserved nucleotide positions within a tRNA acceptor group, but also positions with more than one nucleotide that differ from the corresponding nucleotide positions in other tRNA acceptor groups. In addition, because the between-group variation is considered, the newer method provides information for single tRNA sequences and can perform impressively when only two tRNA isoacceptor sequences are known. The chapter summarizes experiments that define specificity determinants in tRNAs for several amino acids, and includes illustrations of computer predictions. The implications of the study results are discussed, and the chapter closes with an outline of future prospects.

The recognition of a tRNA by its aminoacyl-tRNA synthetase is a classic example of the specificity often encountered in biology. Each of the 20 aminoacyl-tRNA synthetases in a cell must distinguish its own set of isoacceptor tRNAs from the many noncognate tRNAs and efficiently catalyze the covalent attachment of the correct amino acid to the 3' end of only these species. Ultimately, the fate of the cell rests on this interaction, as there are no subsequent proof-reading steps in protein synthesis whereby the amino acid is matched against the anticodon to ensure that the proper amino acid is inserted in response to a given codon. How an aminoacyl-tRNA synthetase is able to select its tRNA substrates from a pool of noncognate species sharing similar tertiary structure has been the focus of over 20 years of research. Recent technical refinements in the types of assays used to study this interaction have contributed a wealth of new information to this field, allowing the identification of nucleotides conferring a particular amino acid acceptor identity for a number of tRNAs. The goal of this chapter is to summarize these more recent developments in tRNA recognition.

This chapter reviews the recognition specificity of a class II aminoacyl-tRNA synthetases (aaRS) and AspRS in detail. Class II aaRS are characterized by three signature motifs, each containing strictly conserved residues. The crystal structures of SerRS from Escherichia coli and AspRS from yeast gave a clear structural explanation for the partition into the two classes. Indeed, class II aaRS contain a new fold, consisting of an antiparallel β sheet flanked by two α helices. The structures of the complex formed by tRNAAsp and AspRS from yeast, with or without ATP correlate these motifs with their biological function; highly conserved residues from motifs 2 and 3 are responsible for ATP binding. Seven class II aaRS exhibit motifs 1 , 2 , and 3. More stringent sequence homology requirements led to the definition of subclasses. Class IIc aaRS (GlyRS, AlaRS, and PheRS) do not contain motif 1 and have a different quaternary organization. PheRS is an even more special case because it has the motif 2 and 3 characteristics of class II but behaves like a class I synthetase as to its primary site of aminoacylation.

This chapter discusses the position and actions of tRNA within the ribosome, a topic that includes some of the principal events of gene expression. It combines critical data to give a consistent picture of tRNA structure and dynamics during coding and chain extension on bacterial ribosomes. The chapter hypothesizes that the stability of the ribosome-bound tRNA, after a conformational change involving its D-anticodon domain (which is called "waggle"), may determine this slowed cognate dissociation rate. Accordingly, the energetics of the somewhat altered tRNA conformation within the ribosome must be considered to predict the outcome of a translational cycle. As one consequence, mutations in "noncoding" nucleotides that alter tRNA conformational preferences appear in genetic selections for coding phenotypes; waggle rationalizes varied genetic data under a single hypothesis. Waggle trades some of the strength of association for greater precision; because it is based on principles that generalize, this trade could be a frequently used strategy for precision in molecular complexes with potentially large interaction energies.

This chapter talks about the genetic translational suppression, that is, suppression caused by a mutation in the gene for one of the translational macromolecules, particularly tRNAs. Such suppressor mutants usually generate a much stronger suppression signal, more easily allowing the analysis of the alteration in translational fidelity. Furthermore, they allow the study of structural determinants of that macromolecule's functions and of its functional interactions with other translational macromolecules. Translational suppression is a most effective way to examine the structure, function, and interactions of any translational macromolecule, as long as and to the extent that that molecule is involved in the specificity or accuracy of translation. The chapter is divided into four parts: (a) review of the requirements of any system to be used for the in vivo selection and study of suppressors, (b) examples of interesting suppressors of missense, nonsense, and frameshift mutations, (c) conclusions from and ramifications of some suppressor tRNA studies, and (d) discussion of some suppressors that are not altered tRNAs but that nevertheless lead, directly or indirectly, to altered functioning of some tRNA.

The occurrence of the amino acid selenocysteine in proteins was first demonstrated for protein A of glycine reductase from Clostridium sticklandii in 1976, and questions were immediately raised on its mechanism of incorporation. At that time, the universality of the 20 proteinogenic amino acids was taken for granted, as was the fact that the 64 codons of the "universal" genetic code are assigned either to code for one of these 20 amino acids or to serve as termination signals. Thus, it seemed unlikely that selenocysteine would be considered as a classical amino acid. In principle, the definition of such a 21st amino acid would require (i) that its incorporation proceeds via a cotranslational mechanism, (ii) that it is directed by a specific codon, and (iii) that a specific tRNA mediates its transport to the ribosome. This chapter illustrates that selenocysteine fulfills these criteria. It first describes the unusual structural properties of tRNASec, and then discusses the unique pathway of selenocysteine insertion that has been worked out for Escherichia coli, which has finally led to the proposal of a model for the co-translational incorporation process at the ribosome. The chapter further compares the pathway established in E. coli with the current knowledge on the mammalian system. Finally, it addresses the interesting question of the evolution of the pathway for the incorporation of selenocysteine that differs from that of the 20 standard amino acids in many respects.