Emergence of the universal genetic code imprinted in an RNA record.
(25/111)
The molecular basis of the genetic code manifests itself in the interaction of the aminoacyl-tRNA synthetases and their cognate tRNAs. The fundamental biological question regarding these enzymes' role in the evolution of the genetic code remains open. Here we probe this question in a system in which the same tRNA species is aminoacylated by two unrelated synthetases. Should this tRNA possess major identity elements common to both enzymes, this would favor a scenario where the aminoacyl-tRNA synthetases evolved in the context of preestablished tRNA identity, i.e., after the universal genetic code emerged. An experimental system is provided by the recently discovered O-phosphoseryl-tRNA synthetase (SepRS), which acylates tRNA(Cys) with phosphoserine (Sep), and the well known cysteinyl-tRNA synthetase, which charges the same tRNA with cysteine. We determined the identity elements of Methanocaldococcus jannaschii tRNA(Cys) in the aminoacylation reaction for the two Methanococcus maripaludis synthetases SepRS (forming Sep-tRNA(Cys)) and cysteinyl-tRNA synthetase (forming Cys-tRNA(Cys)). The major elements, the discriminator base and the three anticodon bases, are shared by both tRNA synthetases. An evolutionary analysis of archaeal, bacterial, and eukaryotic tRNA(Cys) sequences predicted additional SepRS-specific minor identity elements (G37, A47, and A59) and suggested the dominance of vertical inheritance for tRNA(Cys) from a single common ancestor. Transplantation of the identified identity elements into the Escherichia coli tRNA(Gly) scaffold endowed facile phosphoserylation activity on the resulting chimera. Thus, tRNA(Cys) identity is an ancient RNA record that depicts the emergence of the universal genetic code before the evolution of the modern aminoacylation systems. (+info)
Non-enzymatic aminoacylation of an RNA minihelix with an aminoacyl phosphate oligonucleotide.
(26/111)
Efficient aminoacylation of an RNA minihelix (a proposed precursor of tRNA) was achieved without any ribozymes or protein enzymes. The reaction was promoted with a template-like bridging Oligonucleotide and a 5'-aminoacyl-phosphate-oligonucleotide. Aminoacylation was template-dependent and showed high preference for a particular ribose hydroxyl. The 5'-aminoacyl-phosphate-oligonucleotides have the same adenylate linkage as in aminoacyl-AMP--the universal intermediate used in modern aminoacylation systems with aminoacyl-tRNA synthetases. Thus, the results raise the possibility that this kind of system could have played a role in the transition from the RNA world to the theatre of proteins. (+info)
Flexizyme as a versatile tRNA acylation catalyst and the application for translation.
(27/111)
Here we describe a de novo tRNA acylation system consisting of artificial ribozymes. This system, the flexizyme (Fx) system, allows for the preparation of acyl-tRNAs with almost no limitation on the use of a variety of amino/hydroxy acids and tRNAs. To demonstrate its utility, we have carried out protein synthesis involving site-specific incorporation of nonnatural amino and hydroxy acids via amber or programmed frame-shift suppressions. We have also demonstrated peptide synthesis involving multiple nonnatural amino acids via sense codon suppression. The combination of the Fx system and appropriate cell-free translation is a powerful and flexible tool for mRNA-encoded synthesis of nonnatural polypeptides. (+info)
Translation initiation by using various N-acylaminoacyl tRNAs.
(28/111)
Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine. (+info)
Functional association between three archaeal aminoacyl-tRNA synthetases.
(29/111)
Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS. (+info)
Cyclization reaction of peptide fragment ions during multistage collisionally activated decomposition: an inducement to lose internal amino-acid residues.
(30/111)
During characterization of some peptides (linear precursors of the cyclic peptides showing potential to be anticancer drugs) in an ion trap, it was noted that many internal amino acid residues could be lost from singly charged b ions. The phenomenon was not obvious at the first stage of collisionally activated decomposition (CAD), but was apparent at multiple stages of CAD. The unique fragmentation consisting of multiple steps is induced by a cyclization reaction of b ions, the mechanism of which has been probed by experiments of N-acetylation, MS(n), rearranged-ion design, and activation-time adjustment. The fragmentation of synthetic cyclic peptides demonstrates that a cyclic peptide intermediate (CPI) formed by b ion cyclization exhibits the same fragmentation pattern as a protonated cyclic peptide. Although no rules for the cyclization reaction were discerned in the experiments of peptide modification, the fragmentations of a number of b ions indicate that the "Pro and Asn/Gln effects" can influence ring openings of CPIs. In addition, large-scale losses of internal residues from different positions of a-type ions have been observed when pure helium was used as collision gas. The fragmentation is initiated by a cyclization reaction forming an a-type ion CPI. This CPI with a fixed-charge structure cannot be influenced by the "Pro effect", causing a selective ring opening at the amide bond Pro-Xxx rather than Xxx-Pro. With the knowledge of the unique fragmentations leading to internal residue losses, the misidentification of fragments and sequences of peptides may be avoided. (+info)
An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response.
(31/111)
Mistranslation in bacterial and mammalian cells leads to production of statistical proteins that are, in turn, associated with specific cell or animal pathologies, including death of bacterial cells, apoptosis of mammalian cells in culture, and neurodegeneration in the mouse. A major source of mistranslation comes from heritable defects in the editing activities of aminoacyl-tRNA synthetases. These activities clear errors of aminoacylation by deacylation of mischarged tRNAs. We hypothesized that, in addition to previously reported phenotypes in bacterial and mammalian systems, errors of aminoacylation could be mutagenic and lead to disease. As a first step in testing this hypothesis, the effect of an editing defect in a single tRNA synthetase on the accumulation of mutations in aging bacteria was investigated. A striking, statistically significant, enhancement of the mutation rate in aging bacteria was found. This enhancement comes from an increase in error-prone DNA repair through induction of the bacterial SOS response. Thus, mistranslation, as caused by an editing-defective tRNA synthetase, can lead to heritable genetic changes that could, in principle, be linked to disease. (+info)
Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase.
(32/111)
Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS. (+info)