Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems.
ABSTRACT: Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.
Project description:The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of cytosolic, nucleus-encoded tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and the mitochondrion, and all are of the eukaryotic type. This is also the case for the elongator tRNA(Met), whereas the only other trypanosomal tRNA(Met), the eukaryotic initiator, is found exclusively in the cytosol. Unlike their cytosolic counterparts, organellar initiator tRNAs(Met) carry a formylated methionine. This raises the question of how initiation of translation works in trypanosomal mitochondria, where only elongator tRNA(Met) is found. Using in organello charging and formylation assays, we show that unexpectedly a fraction of elongator tRNA(Met) becomes formylated after import into mitochondria. Furthermore, in vitro experiments with mitochondrial extracts demonstrate that only the trypanosomal elongator and not the initiator tRNA(Met) is recognized by the formylation activity. Finally, RNA interference assays identify the gene encoding the trypanosomal formylase activity. Whereas the predicted protein is homologous to prokaryotic and mitochondrial methionyl-tRNA(Met) formyltransferases, it has about twice the mass of any of these proteins.
Project description:In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.
Project description:Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNA(i)) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNA(i) IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNA(i) the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNA(i) conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNA(i). IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.
Project description:Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.
Project description:Activation of amino acid homocysteine was compared with that of methionine in rabbit crude liver extracts and purified multi-enzyme complex of aminoacyl-tRNA synthetases. Activation was studied by measuring the incorporation of radioactive amino acid into unlabelled trichloroacetic-acid insoluble materials in the absence of protein synthesis. Homocysteine synthetase activity was found in the crude extract and in the purified multi-enzyme complex of aminoacyl-tRNA synthetases. On a molar basis, the activation of methionine by the crude extract was five times higher than the activation of homocysteine. There was a partial loss of Hcy-tRNA synthetase activity in the purified multi-enzyme complex. Preliminary reconstitution experiments indicated a requirement for an additional factor for Hcy-tRNA synthetase activity. TLC of the amino acid released from tRNA charged with [14C]homocysteine, revealed radioactivity in homocysteine, methionine and homocysteine thiolactone, indicating a conversion of tRNA-attached homocysteine to methionine. Total tRNA was separated on a benzoylated cellulose column into a fraction enriched in initiator tRNA and a methionine-accepting, but initiator tRNA-deficient, fraction. Homocysteine-accepting activity was present only in the initiator tRNA-enriched fraction. Based on the above data we propose that homocysteine activation in reticulocyte lysates, reported previously, also occurs in liver. Activated homocysteine is attached to initiator tRNA and then converted to methionine by a methylating enzyme. In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone.
Project description:In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5'-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAi(fMet)) while binding of elongator tRNA(Met) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5' met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAi(fMet) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.
Project description:The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its ?-subunit by stress-activated eIF2? kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B-eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.
Project description:All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-I<sub>Xcc</sub>) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-I<sub>Xcc</sub> controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-I<sub>Xcc</sub> expression platform not only can repress gene expression in response to SAM binding to SAM-I<sub>Xcc</sub> aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.
Project description:We have developed three strategies to discriminate among the three types of tRNA genes with anticodon CAT (tRNA(Ile), elongator tRNA(Met) and initiator tRNA(fMet)) in bacterial genomes. With these strategies, we have classified the tRNA genes from 234 bacterial and several organellar genomes. These sequences, in an aligned or unaligned format, may be used for the identification and annotation of tRNA (CAT) genes in other genomes. The first strategy is based on the position of the problem sequences in a phenogram (a tree-like network), the second on the minimum average number of differences against the tRNA sequences of the three types and the third on the search for the highest score value against the profiles of the three types of tRNA genes. The species with the maximum number of tRNA(fMet) and tRNA(Met) was Photobacterium profundum, whereas the genome of one Escherichia coli strain presented the maximum number of tRNA(Ile) (CAT) genes. This last tRNA gene and tilS, encoding an RNA-modifying enzyme, are not essential in bacteria. The acquisition of a tRNA(Ile) (TAT) gene by Mycoplasma mobile has led to the loss of both the tRNA(Ile) (CAT) and the tilS genes. The new tRNA has appropriated the function of decoding AUA codons.
Project description:Mitochondrial (mt) tRNA(Met) has the unusual modified nucleotide 5-formylcytidine (f(5)C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNA(Met) carrying f(5)C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNA(Met) lacking f(5)C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNA(Met)(m) with the anticodon ac(4)CAU (ac(4)C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNA(Met) (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f(5)C modification in mt tRNA(Met) plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f(5)C-G and f(5)C-A based on the chemical properties of f(5)C are presented.