Protein synthesis. Discriminating right from wrong.
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Peptide Biosynthesis↗
Biomedical subjects
M Ibba
Publications and source records attributed to M Ibba.
Protein synthesis: twenty three amino acids and counting.
The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.
Amino Acids↗
Genomics-based identification of targets in pathogenic bacteria for potential therapeutic and diagnostic use.
The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.
Amino Acyl-tRNA Synthetases↗
Adding new meanings to the genetic code.
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Amino Acids↗
The renaissance of aminoacyl-tRNA synthesis.
The role of tRNA as the adaptor in protein synthesis has held an enduring fascination for molecular biologists. Over four decades of study, taking in numerous milestones in molecular biology, led to what was widely held to be a fairly complete picture of how tRNAs and amino acids are paired prior to protein synthesis. However, recent developments in genomics and structural biology have revealed an unexpected array of new enzymes, pathways and mechanisms involved in aminoacyl-tRNA synthesis. As a more complete picture of aminoacyl-tRNA synthesis now begins to emerge, the high degree of evolutionary diversity in this universal and essential process is becoming clearer.
Amino Acids↗
Context-dependent anticodon recognition by class I lysyl-tRNA synthetases.
Lysyl-tRNA synthesis is catalyzed by two unrelated families of aminoacyl-tRNA synthetases. In most bacteria and all eukarya, the known lysyl-tRNA synthetases (LysRSs) are subclass IIb-type aminoacyl-tRNA synthetases, whereas many archaea and a scattering of bacteria contain an unrelated class I-type LysRS. Examination of the recognition of partially modified tRNA(Lys) anticodon variants by a bacterial (from Borrelia burgdorferi) and an archaeal (from Methanococcus maripaludis) class I lysyl-tRNA synthetase revealed differences in the pattern of anticodon recognition between the two enzymes. U35 and U36 were both important for recognition by the B. burgdorferi enzyme, whereas only U36 played a role in recognition by M. maripaludis LysRS. Examination of the phylogenetic distribution of class I LysRSs suggested a correlation between recognition of U35 and U36 and the presence of asparaginyl-tRNA synthetase (AsnRS), which also recognizes U35 and U36 in the anticodon of tRNA(Asn). However, the class II LysRS of Helicobacter pylori, an organism that lacks AsnRS, also recognizes both U35 and U36, indicating that the presence of AsnRS has solely influenced the phylogenetic distribution of class I LysRSs. These data suggest that competition between unrelated aminoacyl-tRNA synthetases for overlapping anticodon sequences is a determinant of the phylogenetic distribution of extant synthetase families. Such patterns of competition also provide a basis for the two separate horizontal gene transfer events hypothesized in the evolution of the class I lysyl-tRNA synthetases.
Anticodon↗
Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding.
The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.
Acylation↗
One polypeptide with two aminoacyl-tRNA synthetase activities.
The genome sequences of certain archaea do not contain recognizable cysteinyl-transfer RNA (tRNA) synthetases, which are essential for messenger RNA-encoded protein synthesis. However, a single cysteinyl-tRNA synthetase activity was detected and purified from one such organism, Methanococcus jannaschii. The amino-terminal sequence of this protein corresponded to the predicted sequence of prolyl-tRNA synthetase. Biochemical and genetic analyses indicated that this archaeal form of prolyl-tRNA synthetase can synthesize both cysteinyl-tRNA(Cys) and prolyl-tRNA(Pro). The ability of one enzyme to provide two aminoacyl-tRNAs for protein synthesis raises questions about concepts of substrate specificity in protein synthesis and may provide insights into the evolutionary origins of this process.
Amino Acyl-tRNA Synthetases↗
The adaptor hypothesis revisited.
As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.
Amino Acyl-tRNA Synthetases↗
Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.
The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.
Amino Acids↗
Aminoacyl-tRNA synthesis.
Aminoacyl-tRNAs are substrates for translation and are pivotal in determining how the genetic code is interpreted as amino acids. The function of aminoacyl-tRNA synthesis is to precisely match amino acids with tRNAs containing the corresponding anticodon. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase, although intrinsic proofreading and extrinsic editing are also essential in several cases. Recent studies of aminoacyl-tRNA synthesis, mainly prompted by the advent of whole genome sequencing and the availability of a vast body of structural data, have led to an expanded and more detailed picture of how aminoacyl-tRNAs are synthesized. This article reviews current knowledge of the biochemical, structural, and evolutionary facets of aminoacyl-tRNA synthesis.
Animals↗
Quality control mechanisms during translation.
Translation uses the genetic information in messenger RNA (mRNA) to synthesize proteins. Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. The cycle is then repeated to produce a full-length protein. Proofreading and editing processes are used throughout protein synthesis to ensure the faithful translation of genetic information. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base pairing with mRNA. Recent studies have begun to reveal the molecular mechanisms underpinning quality control and go some way to explaining the phenomenal accuracy of translation first observed over three decades ago.
Amino Acyl-tRNA Synthetases↗
Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.
Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.
Adenosine Monophosphate↗
tRNA recognition and evolution of determinants in seryl-tRNA synthesis.
We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.
Acylation↗
Substrate recognition by class I lysyl-tRNA synthetases: a molecular basis for gene displacement.
Lysyl-tRNA synthetases (LysRSs) are unique amongst the aminoacyl-tRNA synthetases in being composed of unrelated class I and class II enzymes. To allow direct comparison between the two types of LysRS, substrate recognition by class I LysRSs was examined. Genes encoding both an archaeal and a bacterial class I enzyme were able to rescue an Escherichia coli strain deficient in LysRS, indicating their ability to functionally substitute for a class II LysRS in vivo. In vitro characterization showed lysine activation and recognition to be tRNA-dependent, an attribute of several class I, but not class II, aminoacyl-tRNA synthetases. Examination of tRNA recognition showed that class I LysRSs recognize the same elements in tRNALys as their class II counterparts, namely the discriminator base (N73) and the anticodon. This sequence-specific recognition of the same nucleotides in tRNALys by the two unrelated types of enzyme suggests that tRNALys predates at least one of the LysRSs in the evolution of the translational apparatus. The only observed variation in recognition was that the G2.U71 wobble pair of spirochete tRNALys acts as antideterminant for class II LysRS but does not alter class I enzyme recognition. This difference in tRNA recognition strongly favors the use of a class I-type enzyme to aminoacylate particular tRNALys species and provides a molecular basis for the observed displacement of class II by class I LysRSs in certain bacteria.
Amino Acyl-tRNA Synthetases↗
Archaeal aminoacyl-tRNA synthesis: diversity replaces dogma.
Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not.
Amino Acyl-tRNA Synthetases↗
Archaeal aminoacyl-tRNA synthesis: unique determinants of a universal genetic code?
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Archaea↗