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Category: Microbial Genetics and Molecular Biology
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The rapid progress of research in the tRNA field and recent advances in the understanding of the molecular basis of specificity in tRNA: protein interactions make it necessary to have all of the accumulated information in an easily accessible form. The purpose of this book is to fulfill that need by providing an up-to-date account of all aspects of research on transfer RNA, including its structure, biosynthesis, and interactions with the many proteins involved in protein biosynthesis.
Beginning with an historical account, the book covers a broad area of research on tRNA biosynthesis, the different functions of tRNA in the genetic decoding process, its association with many different proteins, and the emerging rules governing the specificity of their interactions. In view of the impressive progress made in the last few years, several of the chapters are devoted to discussion of aminoacyl-tRNA synthetase tRNA interactions. An appendix containing the structural formulae of all modified nucleosides found in tRNA completes this book.
Electronic only, 580 pages, index.
Nearly four decades have passed since the discovery of tRNA. Much progress was made in the first two decades: elucidation of the role of tRNA as an adaptor in protein biosynthesis, determination of the primary sequences of several tRNAs, and solution of the three-dimensional structure of a tRNA. In addition, genes for tRNA were synthesized and the synthetic gene for an amber suppressor tRNA was shown to be functional in vivo. Progress has been made on a broad front in the last two decades also, and tRNA has remained an attractive and active area of research. In summarizing the four decades of tRNA research, it is appropriate to note that tRNA has been the source of many firsts in nucleic acid research.
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 presents a summary of information on the structures and organization of prokaryotic tRNA genes based on recent work surveying the total tRNA populations and tRNA genes in Escherichia coli by Ozeki’s group at Kyoto and in Mycoplasma capricolum by Osawa’s group at Nagoya. Most importantly, E. coli and M. capricolum are the only cases to date in which the total tRNA systems have been well elucidated, including the sequences of a complete set of tRNAs, the organization and expression of their genes, composition of the full anticodon set, and the relative levels of each tRNA species in the cells. In general, tRNA genes are found in clusters. For example, among 79 tRNA genes of E. coli, only 20 exist as a single gene. The remaining 59 are organized in polycistronic operons containing identical or unrelated tRNA, rRNA genes, and protein-encoding genes. In M. capricolum, 22 of 30 tRNA genes are organized in five clusters comprising nine, five, four, and two genes, respectively. The other eight genes exist as single transcription units. In summary, the decoding system of M. capricolum is significantly different from that of E. coli in the number of anticodons, codon recognition patterns, and, in two cases, codon assignment.
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 focuses on ribonucleases that are involved in generating the 3' terminus of tRNA precursors and in cleaving a tRNA from a multicomponent transcript. Much of our knowledge about tRNA-processing pathways has come from studies with Escherichia coli and bacteriophage-infected E. coli, although there has been some examination of other bacteria as well. The major interest in E. coli is due primarily to the availability, in this system, of mutations that interfere with tRNA maturation, allowing processing intermediates to be identified and processing nucleases to be implicated in the processing pathway. However, the tRNA precursors isolated from these mutant strains have undergone partial processing. First, the tRNA portions of polycistronic precursors have been cleaved from the other RNAs with which they are co-transcribed. Second, multimeric tRNA precursors generally have been converted to monomeric or dimeric forms. A major goal in the study of any metabolic pathway is to identify the enzymes that catalyze each of its reactions, to understand their specificity and mechanism of action, and to determine whether they are subject to regulation, either directly on their activity or on their synthesis. The chapter presents a summary of the exoribonucleases and endoribonucleases implicated in tRNA processing. Given the availability of mutant strains lacking many of these enzymes, alone or in combination, it is likely that the details of a tRNA processing pathway will be forthcoming in the near future.
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 reviews what is known about the mechanism of precursor tRNA splicing: (i) the tRNA substrates for the splicing reaction, (ii) the enzymes involved in removing the introns to form the mature tRNA, (iii) interactions between these enzymes and their tRNA substrates and cofactors, (iv) the organization of tRNA splicing in the nucleus, (v) the identity of splicing mutants that affect the enzymatic machinery, (vi) current knowledge about the differences and similarities of tRNA splicing in systems of various organisms, and (vii) the possible function of tRNA introns.
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.
Mitochondria and chloroplasts contain their own systems for the biosynthesis of proteins coded by the organelle genome. It had generally been thought that tRNAs participating in organelle protein synthesis were coded by the organelle genome. This genetic diversity of tRNAs makes the subject of organelle tRNA biosynthesis more complex than it would be if all tRNAs were made in the organelle. tRNA genes in organelle DNAs are scattered around the genome and intermixed with mRNA and rRNA genes. tRNAs are transcribed with rRNAs and mRNAs. In mammalian mitochondria, the entire genome is transcribed and, subsequently, the tRNAs are processed out of the large primary transcript. This review focuses on the organelle tRNAs that are coded by mitochondrial or chloroplast DNA and on the structure of precursor tRNAs and the enzymes necessary for their processing. In addition, it considers organelle aminoacyl-tRNA synthetases as well as mutant organelle tRNAs.
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.
Modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U), were found in nucleic acids as early as 1948. Modified nucleosides are contained in tRNA from all three phylogenetic domains—Archaea, Bacteria, and Eucarya—which were formerly called the kingdoms of archaebacteria, eubacteria, and eukaryotes, respectively. Although modified nucleosides are found in various positions in the tRNA, two positions, 34 and 37, contain the largest variety of modified nucleosides. This chapter presents an overview of the coding properties associated with modified nucleosides present in positions 34 and 37. Outside the anticodon, the modified nucleosides are usually “simple” modifications like methylated or thiolated derivatives, whereas all except one (archaeosine in tRNAs from Archaea) of the hypermodified nucleosides are present in the anticodon region and only in positions 34 and 37. Moreover, outside the anticodon region, only one or two kinds of modified nucleosides in each position are present, whereas a large variety of modified nucleosides are present in the anticodon region, especially in positions 34 and 37. During evolution, structural refinements of the individual tRNAs were presumably fulfilled by the evolution of the synthesis of the various modified nucleosides, including the hypermodified nucleoside. Modified nucleosides in the anticodon region exert their functions primarily in the decoding process, whereas modified nucleosides outside this region may primarily be involved in other tRNA interactions, such as interactions with translation factors or as sensors for environmental stress conditions.
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.
This chapter reviews the recent status of variations in the genetic code in various mitochondrial and nuclear systems. It explains relationship between the reading patterns of the changed codons and the anticodons of the tRNAs involved. It also considers mRNA editing in plant and protozoan organelles and a revision of codon assignments on the basis of protein gene sequences in the universal code, which is based on mRNA sequences for the proteins.
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.
Aminoacyl-tRNA synthetases (AARS) form a class of essential enzymes whose main role is to ligate amino acids to tRNAs. This chapter reviews what is known about the AARS genes, their chromosomal localization, their organization, and the regulation of their expression. It focuses exclusively on bacterial systems, mainly Escherichia coliand Bacillus subtilis, for which a great deal of information is available.
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.
Transfer RNAs fold into a globular two-domain, L-shaped structure with the amino acid acceptor terminus and anticodon at opposite ends. This chapter reports nine examples of sequence-specific aminoacylation of RNA oligonucleotides based on tRNA acceptor stems with both class I and class II tRNA synthetases. The examples are class I aminoacyl-tRNA synthetases for Met, He, Gin, and Val and class II aminoacyl-tRNA synthetases for Ala, His, Asp, Ser, and Gly. In these examples, the aminoacylation activity for RNA oligonucleotide substrates is commonly greater for the class II enzymes. The exception is the class I He tRNA synthetase. This variation in activity may be due to the difference in the way the 3' end of tRNA interacts with the class I enzymes compared with the class II enzymes. The structures of the class I Gln-tRNA synthetase-tRNAGln complex and the class II Asp-tRNA synthetase-tRNAAsP complex indicate that the binding of the 3' end of the tRNA is fundamentally different. For example, interactions between the minor groove of the acceptor stem of tRNAGln and Gln tRNA synthetase disrupt the first base pair of the tRNA and induce a hairpin turn of the 3' terminus toward the inside of the L-shaped tRNA. The relationship between the attached amino acids and the sequences of RNA oligonucleotide substrates constitute an operational RNA code for amino acids.
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.
The ability of aminoacyl-tRNA synthetases (aaRS) to faithfully recognize both their amino acid and tRNA substrates is essential for accurate protein synthesis. This chapter focuses on the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). This system is arguably the best characterized aaRS-tRNA interaction both functionally and structurally, as the first high-resolution crystal structure of a protein-RNA complex was solved for GlnRS:tRNAGln. The chapter summarizes the characteristics of the GlnRS:tRNAGln system that make GlnRS unique among aaRS, as well as those that make the glutamine system an ideal model for the study of protein-RNA interaction, specifically aaRS-tRNA interaction. Together the crystal structure, genetic, and biochemical studies have identified the most important specificity determinants from among the hundreds of contacts between GlnRS and tRNAGln. The chapter also describes several of the more interesting aspects of the GlnRS:tRNAGln system: (1) the close evolutionary relationship between GlnRS and glutamyl-tRNA synthetase (GluRS); (2) GlnRS's relaxed discrimination against noncognate tRNAs coupled with its "overdetermined," tight recognition of its cognate tRNA; and (3) the enzyme mechanism of GlnRS, specifically the structural and functional communication that permits this small monomeric aaRS to recognize tRNA identity elements that are more than 75Å apart in uncomplexed tRNA. The information obtained from biophysical techniques (crystallography, fluorescence, and x-ray/neutron scattering) and from genetic and biochemical approaches is combined to yield a coherent and detailed picture of the specific recognition of tRNA by E. coli GlnRS.
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.
The interactive recognition of nucleic acids by proteins is a central process in the regulation of gene expression. To gain insight into such interactions requires a knowledge of appropriate three-dimensional structures and information on biochemical function. Only a few native biological supramacromolecular protein-nucleic acid complexes are currently accessible for such detailed investigations. One convenient object for such study is the ternary complex composed of aminoacyl-tRNA (aa-tRNA) and elongation factor (EF-Tu) bound to GTR. This chapter brings up to date two earlier reviews addressing the problem of aa-tRNA and EF-Tu:GTP interaction. It summarizes the most recent published studies that contribute to an understanding of the recognitory interactions between tRNA and the protein elongation factor, and the chapter puts these studies in perspective. Finally, it proposes a new model for the three-dimensional structure of the ternary complex (TC). This model accommodates all existing structural experimental data obtained during the studies of the TC.
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.
Virtually all tRNAs are known or predicted from their DNA sequence to have seven-base anticodon loops. If natural, functional tRNAs with eight or nine-base anticodon loops exist, they may cause frameshifting, like their mutant counterparts, whether or not they also participate in triplet decoding. Recoding due to re-pairing has only recently been discovered, and it is certain that surprises lie ahead. Nevertheless, some tentative generalization of the available data on re-pairing at overlapping sites seems warranted. The +1 frameshifting uses a single shift site and offers the opportunity for regulation. In at least one case, regulation works through competition for the downstream codon, so as to sense some biochemical state. Even when the downstream codon is not used for this purpose, tandem shift codons are not used. In contrast, efficient —1 frameshifting uses tandem shift sites. Regulation is not seen, and both pre-slip A and P sites are occupied by tRNA generating the efficient double shift mechanism. Using this mechanism, weak pre-slip A site pairing seems to be important, although there is surprising latitude in repairing. An interesting issue is whether the absence to date of tandem +1 shifts is fortuitous and, if not, the reason for their absence. Several cases of programed frameshifting use more than one stimulatory signal. The +1 frameshifting often utilizes a 5' stimulator, whereas efficient —1 frameshifting often uses a stimulator 3' to the site.
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.
Initiation of protein synthesis occurs universally with the amino acid methionine or its derivative formyl methionine. Of the two classes of methionine tRNAs present in all organisms, the initiator is used for initiation of protein synthesis, whereas the elongator is used for insertion of methionine into internal peptidic linkages. In eubacteria and in eukaryotic organelles such as chloroplasts and mitochondria, the initiator tRNAs are used as formylmethionyl-tRNA (fMet-tRNA). In the cytoplasmic protein synthesis system of eukaryotes and in archaebacteria, they are used as methionyl-tRNA (Met-tRNA) without formylation. This chapter focuses on initiator tRNAs and their role in initiation of protein synthesis. It provides a brief and somewhat simplified description of some of the steps of protein synthesis initiation that involve the initiator tRNA most directly. Then, it describes the special properties of eubacterial and eukaryotic initiator tRNAs and the current knowledge of the relationship between the sequence and structure of the initiator tRNAs and their function.
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.
Since the discovery of initiator tRNA, a number of reactions have been found that involve modification of amino acids attached to tRNA. These reactions include well-understood processes like the formation of formylmethionyl-tRNA, which serves to initiate peptide chain formation in prokaryotes, or the synthesis of selenocysteine, which functions as the 21st amino acid. Less well understood are the conversions of glutamate. The first conversion serves for the formation of glutamate-1-semialdehyde, the initial precursor of porphyrins (e.g., chlorophylls and hemes). The other known reaction is a transamidation of glutamate attached to tRNAGln, which is a required intermediate in the formation of glutaminyl-tRNA in many organisms and organelles. These conversions of glutamate based on Glu-tRNA as intermediate are the topic of this chapter. Tetrapyrrole-containing compounds, such as hemes and chlorophylls, are essential components of respiratory and photosynthetic reactions. The porphyrin ring of these compounds is derived from eight molecules of 5-aminolevulinic acid (ALA), a precursor whose formation provides a key regulatory control point in heme and chlorophyll biosynthesis.
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