Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.13.12.7 (firefly luciferase)
 2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.
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PMID:Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase. 176 Oct 51

The synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl-tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275-8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959-2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E-aminoacyl-AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E-luciferin-AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl-containing intermediates and releasing PPi may produce dinucleoside polyphosphates.
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PMID:Firefly luciferase synthesizes P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates. 217 2

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.
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PMID:L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. 762 62

A nuclear tRNA(Lys) gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA(Lys) and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA(LysCUA) from non-replicative vectors promoted 10-40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA(Lys) suppressor genes from a geminivirus vector capable of replication promoted 30-80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.
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PMID:Nonsense and missense translational suppression in plant cells mediated by tRNA(Lys). 948 71

The Arg attenuator peptide (AAP) is an evolutionarily conserved peptide involved in Arg-specific negative translational control. It is encoded as an upstream open reading frame (uORF) in fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase. We examined the functions of the Saccharomyces cerevisiae CPA1 and Neurospora crassa arg-2 AAPs using translation extracts from S. cerevisiae, N. crassa, and wheat germ. Synthetic RNA containing AAP and firefly luciferase (LUC) sequences were used to program translation; analyses of LUC activity indicated that the AAPs conferred Arg-specific negative regulation in each system. The AAPs functioned either as uORFs or fused in-frame at the N terminus of LUC. Mutant AAPs lacking function in vivo did not function in vitro. Therefore, trans-acting factors conferring AAP-mediated regulation are in both fungal and plant systems. Analyses of ribosome stalling in the fungal extracts by primer extension inhibition (toeprint) assays showed that these AAPs acted similarly to stall ribosomes in the region immediately distal to the AAP coding region in response to Arg. The regulatory effect increased as the Arg concentration increased; all of the arginyl-tRNAs examined appeared maximally charged at low Arg concentrations. Therefore, AAP-mediated Arg-specific regulation appeared independent of the charging status of arginyl-tRNA.
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PMID:A highly conserved mechanism of regulated ribosome stalling mediated by fungal arginine attenuator peptides that appears independent of the charging status of arginyl-tRNAs. 1060 10

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.
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PMID:Neurospora crassa supersuppressor mutants are amber codon-specific. 1213 72

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.
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PMID:Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase. 1282 Jul 96

Site-specific insertion of modified amino acids in proteins expressed in living cells is an emerging field holding great promise for elucidating protein structure-function relationships, expression levels, localization, and activation states in a complex milieu. To evaluate the efficiency of amino acids modified to carry either a nitroxide spin probe or a fluorescence probe, we have developed a screen using the levels of functional luciferase protein expressed in Xenopus oocytes. Natural and modified amino acids were targeted to position 14 in firefly luciferase using an amber mutation or introducing the four-codon nucleotide GGGU. Using the amber stop codon, the incorporation efficiencies of injected tRNA charged with the native phenylalanine residue, a fluorescent NBD-alanine, or nitroxide-labeled cysteine and tyrosine amino acids ranged from 1% to 18%. While the NBD-amino acid derivative gave higher incorporation levels, the EPR signals from the spin-labeled amino acids allow for the direct assessment of aminoacylation extent and stability. Applying the four-base codon for the first time in Xenopus oocytes, we found the incorporation efficiencies were significantly lowered compared to results using the three-base amber codon. The studies presented here provide quantitative assessment of protein expression levels when using nonsense suppression to site-specifically label proteins with spectroscopic probes in oocytes. Finally, the effect of a 77-base RNA aptamer known to inhibit the eucaryotic release factor of protein synthesis was tested for its influence on nonsense incorporation in Xenopus oocytes. The combination of A34 and charged suppressor tRNA produced a 3-fold increase in the expressed TAG(14)-luciferase level, compared to the use of charged suppressor tRNA alone.
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PMID:Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes. 1522 58

HIV-1 uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes. This frameshift occurs at a specific slippery sequence followed by a stimulatory signal, which was recently shown to be a two-stem helix, for which a three-purine bulge separates the upper and lower stems. In the present study, we investigated the response of the bacterial ribosome to this signal, using a translation system specialized for the expression of a firefly luciferase reporter. The HIV-1 frameshift region was inserted at the beginning of the coding sequence of the luciferase gene, such that its expression requires a -1 frameshift. Mutations that disrupt the upper or the lower stem of the frameshift stimulatory signal or replace the purine bulge with pyrimidines decreased the frameshift efficiency, whereas compensatory mutations that re-form both stems restored the frame-shift efficiency to near wild-type level. These mutations had the same effect in a eukaryotic translation system, which shows that the bacterial ribosome responds like the eukaryote ribosome to the HIV-1 frameshift stimulatory signal. Also, we observed, in contrast to a previous report, that a stop codon immediately 3' to the slippery sequence does not decrease the frameshift efficiency, ruling out a proposal that the frameshift involves the deacylated-tRNA and the peptidyl-tRNA in the E and P sites of the ribosome, rather than the peptidyl-tRNA and the aminoacyl-tRNA in the P and A sites, as commonly assumed. Finally, mutations in 16S ribosomal RNA that facilitate the accommodation of the incoming aminoacyl-tRNA in the A site decreased the frameshift efficiency, which supports a previous suggestion that the frameshift occurs when the aminoacyl-tRNA occupies the A/T entry site.
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PMID:A reassessment of the response of the bacterial ribosome to the frameshift stimulatory signal of the human immunodeficiency virus type 1. 1524 29

Studies employing mRNA transfection are currently limited by a lack of transcription vectors for generating a long poly(A) tail-containing mRNA and published methods for efficient mRNA transfection. We have constructed a transcription vector containing firefly luciferase gene (pBS-FLuc-A100) to generate luciferase mRNA with A100 tail followed by no heterologous sequence. The pBS-FLuc-A100 was propagated in XL1-Blue, in which the plasmid was more stable than in other bacterial strains. Optimal mRNA transfection conditions were determined using TransMessenger Transfection Reagent (Qiagen) and yeast tRNA as a carrier. Firefly luciferase expression, which peaked at about 12 h post-transfection, was detected with as little as 5 ng mRNA and was linear with mRNA amount up to 100 ng. When cells were transfected with luciferase mRNA containing different lengths of poly(A) tail, luciferase expression increased proportionally with poly(A) tail length up to 60A residues and then declined. Cell lines from monkey, mouse, and rat were transfected efficiently by this method. Like cellular ferritin heavy chain mRNA, which contains an iron response element in its 5'UTR, translation of transfected luciferase mRNA containing the 5'UTR of ferritin mRNA was iron-dependent. Our results demonstrate that the poly(A) vector and the transcription method described will be useful to study the regulation of gene expression at the mRNA level by UTRs.
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PMID:Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector. 1580 89


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