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Query: UNIPROT:P06889 (Mol)
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A 16,226-bp fragment from the genome of Aquifex pyrophilus was sequenced, containing the genes for ribosomal proteins L1, L10, and L7/12 (rplAJL), DNA-directed RNA polymerase subunits beta and beta' (rpoBC), alanyl-tRNA synthetase (alaS), and subunit A of proteinase Clp (clpA). Enzymatic activity and extreme thermostability of purified A. pyrophilus RNA polymerase were verified. Transcription initiation on a DNA construct harboring the T7 A1 promoter was demonstrated by elongation of a 32P-labeled trinucleotide. Phylogenetic analyses of the two largest subunits of bacterial RNA polymerases (beta and beta') showed overall consistency with the 16S rRNA-based phylogeny, except for the positions of the hyperthermophiles A. pyrophilus and Thermotoga maritima and for the location of the root of the domain Bacteria. In the phylogenies for both RNA polymerase subunits beta and beta', A. pyrophilus was placed within the Gram-negative bacteria below the epsilon subdivision of the Proteobacteria. No support was found for the 16S rRNA-based hypothesis that A. pyrophilus might be the deepest branch of the Bacteria, but the cell wall-less mycoplasmas were found with a high confidence at the root of the Bacteria phylogenies. This raised doubts not only about whether the original Bacteria were indeed like the hyperthermophiles, but also concerning the value of single-gene phylogenies for hypotheses about the evolution of organisms.
J Mol Evol 1999 May
PMID:RNA polymerase of Aquifex pyrophilus: implications for the evolution of the bacterial rpoBC operon and extremely thermophilic bacteria. 1019 19

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.
J Mol Biol 2001 Nov 16
PMID:Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA. 1172 28

Transfer-messenger RNA (tmRNA), also known as SsrA or 10Sa RNA, is a bacterial ribonucleic acid that recycles 70S ribosomes stalled on problematic messenger RNAs (mRNAs) and also contributes to the degradation of incompletely synthesized peptides. tmRNA acts initially as transfer RNA (tRNA), being aminoacylated at its 3'-end by alanyl-tRNA synthetase, to add alanine to the stalled polypeptide chain. Resumption of translation ensues not on the mRNA on which the ribosomes were stalled but at an internal position in tmRNA. Termination soon occurs, tmRNA recruiting the appropriate termination factors allowing the release of the tagged protein that is subsequently recognized and degraded by specific cytoplasmic and periplasmic proteases, and permits ribosome recycling. Recent data suggest that tmRNA tags bacterial proteins in three other instances; when ribosomes stall at internal sites; during 'readthrough' of canonical termination codons; and when ribosomes are at the termination codon of intact messages. The importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is also discussed. Recent in vivo and in vitro studies have identified novel ligands of tmRNA. Based on the available experimental evidences, an updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented.
Mol Microbiol 2001 Nov
PMID:Emerging views on tmRNA-mediated protein tagging and ribosome rescue. 1173 33

Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.
Mol Cell 2004 Mar 26
PMID:Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition. 1505 76

The crystal structure of a catalytic fragment of Aquifex aeolicus AlaRS and additional data suggest how the critical G3:U70 identity element of its cognate tRNA acceptor stem is recognized. Though this identity element is conserved from bacteria to the cytoplasm of eukaryotes, Drosophila melanogaster mitochondrial (Dm mt) tRNA(Ala) contains a G:U base pair that has been translocated to the adjacent 2:71 position. This G2:U71 is the major determinant for identity of Dm mt tRNA(Ala). Sequence alignments showed that Dm mt AlaRS is differentiated from G3:U70-recognizing AlaRSs by an insertion of 27 amino acids in the region of the protein that contacts the acceptor stem. Precise deletion of this insertion from Dm mt AlaRS gave preferential recognition to a G3:U70-containing substrate. Larger or smaller deletions were ineffective. The crystal structure of the orthologous A. aeolicus protein places this insertion on the surface, where it can act as a hinge that provides positional switching of G:U recognition.
Mol Cell 2004 Mar 26
PMID:Positional recognition of a tRNA determinant dependent on a peptide insertion. 1505 77

Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence of split genes in the Nanoarchaeum genome is a derived feature.
Mol Biol Evol 2005 Jan
PMID:Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids. 1535 78

Previous studies have shown that in Saccharomyces cerevisiae the mitochondrial and cytoplasmic forms of alanyl-tRNA synthetase are encoded by a single nuclear gene, ALA1, through alternative use of in-frame successive ACG triplets and a downstream AUG triplet. Here we show that despite the obvious participation of the non-AUG-initiated leader peptide in mitochondrial localization, the leader peptide per se cannot target a cytoplasmic passenger protein into mitochondria under normal conditions. Functional mapping further shows that an efficient targeting signal is composed of the leader peptide and an 18-residue sequence downstream of Met1. Consistent to this observation, overexpression of the cytoplasmic form enables it to overcome the compartmental barrier and function in the mitochondria as well, but deletion of as few as eight amino acid residues from its amino-terminus eliminates such a potential. Thus, the sequence upstream of the first in-frame AUG initiator not only carries an unusual initiation site, but also contributes to a novel pattern of protein expression and localization.
Mol Microbiol 2006 Apr
PMID:An unusual pattern of protein expression and localization of yeast alanyl-tRNA synthetase isoforms. 1655 30

Aminoacyl-tRNA synthetases are divided into two classes based on both functional and structural criteria. Distinctions between the classes have heretofore been based on general features, such as the position of aminoacylation on the 3'-terminal tRNA ribose, and the topology and tRNA-binding orientation of the active-site protein fold. Here we show instead that transient burst kinetics provides a distinct mechanistic signature dividing the two classes of tRNA synthetases, and that this distinction has significant downstream effects on protein synthesis. Steady-state and transient kinetic analyses of class I CysRS and ValRS, and class II AlaRS and ProRS, reveal that class I tRNA synthetases are rate-limited by release of aminoacyl-tRNA, while class II synthetases are limited by a step prior to aminoacyl transfer. The tight aminoacyl-tRNA product binding by class I enzymes correlates with the ability of EF-Tu to form a ternary complex with class I but not class II synthetases, and the further capacity of this protein to enhance the rate of aminoacylation by class I synthetases. These results emphasize that the distinct mechanistic signatures of class I versus class II tRNA synthetases ensure rapid turnover of aminoacyl-tRNAs during protein synthesis.
J Mol Biol 2006 Aug 11
PMID:Distinct kinetic mechanisms of the two classes of Aminoacyl-tRNA synthetases. 1684 87

Bombardment of a highly expressed dicot tRNA(ala)(GAC) gene into Zea mays bz-E2 or bz-E5 coleoptiles causes suppression of an Ala(458 )-->Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA(ala)(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA(ala)(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.
Plant Mol Biol 2006 Jul
PMID:Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization. 1689 93

The character compatibility approach, which removes all homoplasic characters and involves finding the largest clique of compatible characters in a dataset, in principle, provides a powerful means for obtaining correct topology in difficult to resolve cases. However, the usefulness of this approach to generalized molecular sequence data for phylogeny determination has not been studied in the past. We have used this approach to determine the topology of 23 proteobacterial species (6 each of alpha-, beta- and gamma-, 3 delta-, and 2 epsilon-proteobacteria) using sequence data for 10 conserved proteins (Hsp60, Hsp70, EF-Tu, EF-G, alanyl-tRNA synthetase, RecA, GyrA, GyrB, RpoB and RpoC). All sites in the sequence alignments of these proteins where only two amino acids were found, with each amino acid present in at least two species, were selected. Mutual compatibility determination on these binary state sites was carried out by two means. In one case, all of these sites were combined into a large dataset (Set A; 957 characters) prior to compatibility analysis. In the second case, compatibility analysis was carried out on characters from individual proteins and all compatible sites were combined into a large dataset (Set B; 398 characters) for further studies. Upon compatibility analyses, the largest cliques that were obtained from Sets A and B consisted of 337 and 323 compatible characters, respectively. In these cliques, all proteobacterial subgroups were clearly distinguished and branching orders of most of the species were also resolved. The epsilon-proteobacteria exhibited the earliest branching, whereas the beta- and gamma-subgroups were found to have emerged last. The relative placement of the alpha- and delta-subgroups, however, was not resolved. The topology of these species was also determined based on 16S rRNA sequences and a concatenated dataset of sequences for all 10 proteins by means of neighbor-joining, maximum likelihood, and maximum parsimony methods. In the protein trees, all proteobacterial groups were reliably resolved and they branched in the following order: (epsilon(delta(alpha(beta,gamma)))). However, in the rRNA trees, the gamma- and beta-subgroups exhibited polyphyletic branching and many internal nodes were not resolved. These results indicate that the character compatibility analysis using generalized molecular sequence data provides a powerful means for evolutionary studies. Based on molecular sequences, it should be possible to obtain very large datasets of compatible characters that should prove very helpful in clarifying difficult to resolve phylogenetic relationships.
J Mol Evol 2007 Jan
PMID:Application of the character compatibility approach to generalized molecular sequence data: branching order of the proteobacterial subdivisions. 1716 Jun 41


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