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M Springer

Publications and source records attributed to M Springer.

At least 91 records · Page 5Linked to original sources
PubMed1983

Organization of the Escherichia coli chromosome around the genes for translation initiation factor IF2 (infB) and a transcription termination factor (nusA).

The genes infB, for translational initiation factor IF2, and nusA for a protein involved in transcription termination are carried on a 4.8 X 10(3) base-pair DNA fragment. This fragment also carries promoters capable of expressing both genes. The order of these genes with respect to the surrounding genes is pnp, rpsO, infB, nusA, argG. Transcription of the two genes is anticlockwise on the standard Escherichia coli map, i.e. directed towards the genes rpsO and pnp. The presence of infB on multicopy plasmids enhances IF2 protein and messenger RNA levels only two to threefold compared to the normal haploid level.

Bacterial Proteins↗
PubMed1983

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants.

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Structural and transcriptional evidence for related thrS and infC expression.

The nucleotide sequence of thrS, the gene encoding dimeric Escherichia coli threonyl-tRNA synthetase [L-threonine:tRNAThr ligase (AMP forming), 6.1.1.3], has been determined. The structural part of the gene is found upstream of and on the same DNA coding strand as infC, the gene for translational initiation factor IF3. thrS is composed of 1,926 base pairs and accounts for a protein of molecular weight 73,906. In addition, a 336-base-pair sequence 5' to the thrS structural gene has been determined. There are only three nucleotides between the stop codon of thrS and the initiator codon of infC. The only potent transcriptional terminator structure is 55 base pairs downstream of the infC coding sequence. This implies that thrS and infC can be expressed from a polycistronic mRNA originating from a promoter upstream to thrS. Although sequence data indicate that thrS and infC are cotranscribed, in vitro transcription and RNA sequence analyses reveal the existence of a promoter within thrS. This promoter can account for the independent expression of infC as reported [Springer, M., Plumbridge, J. A., Trudel, M., Graffe, M. & Grunberg-Manago, M. (1982) Mol. Gen. Genet. 186, 247-252]. A second promoter has been located within infC and could link the expression of infC and that of the next downstream gene, pdzA. Whether these promoters function normally in vivo is an open question.

Amino Acid Sequence↗
PubMed1982

Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon.

The nucleotide sequence of a 1.26-kb pair DNA fragment containing the structural gene for Escherichia coli initiation factor IF3 has been determined. An open reading frame of 540 nucleotides is found at the position predicted by genetic studies. The amino-acid sequence deduced from the DNA sequence accounts for a molecular weight 20 530. The important feature of the coding DNA sequence is the presence of AUU as the translational initiator codon. It is 11 bases downstream of the center of a GGAGG sequence, which can strongly pair with the sequence CCUCC near the 3' terminus of 16S rRNA. The primary DNA sequence in the region of the AUU initiator codon and its role in compensating a reduced codon-anticodon interaction in initiation complex formation are discussed.

Amino Acid Sequence↗
PubMed1982

Transcription units around the gene for E. coli translation initiation factor IF3 (infC).

The coding properties have been analyzed of in vitro constructed lambda recombinant phages carrying E. coli DNA fragments from around the structural gene for translation initiation factor IF3 (infC). This study shows that infC is expressed independently of the promoter of the threonyl-tRNA synthetase (thrS), which is the genes immediately preceding infC. It also shows that the two genes following infC, namely pheS and pheT, which form the phenylalanyl-tRNA synthetase operon, are not expressed from infC's promoter. Thus the four characterized genes of that region that were previously thought to be transcribed in the same direction are now shown to be expressed in vivo as three separate transcription units.

DNA, Bacterial↗
PubMed1982

Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli.

A novel method, not relying on genetic complementation of a mutation, was used to clone a gene for translational initiation factor IF2. Two clones from a cosmid library of total Escherichia coli DNA were isolated for their ability to overproduce IF2 in vivo as determined by quantitative immunoblotting. "Maxicell" analysis of cosmid-encoded proteins and specific immune precipitation of the labeled proteins showed that the structural gene for IF2 (inf B) had been cloned. Subcloning fragments from the original cosmids located the inf B gene to a 4.8-kilobase pair HindIII/BamHI fragment. This fragment has been inserted into an integration-deficient recombinant lambda phage that lysogenizes by homology. By mapping the point of lysogenization on the E. coli chromosome, inf B has been located at 68 min, very close to argG, nusA, rpsO, and pnp. Because the gene for initiation factor IF3 is located at 38 min on the chromosome, the genes for translational initiation factors are not grouped together.

Bacteriophage lambda↗
PubMed1982

Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro.

Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene. When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed. IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased. The results suggest that IF-3 alpha is slowly degraded to the beta form. Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis. Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate.

Bacterial Proteins↗
PubMed1982

Escherichia coli phenylalanyl-tRNA synthetase operon: characterization of mutations isolated on multicopy plasmids.

Plasmid pB1 carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying this plasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneous mutant plasmids were obtained which no longer caused the overproduction of the enzyme. Three classes of mutations were found. (i) Deletion mutations were found, some of which had the interesting property of fusing different genes together, e.g., putting phenylalanyl-tRNA synthetase under the control of the threonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; one insertion in particular was studied. This insertion is located in front of the structural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNA structure were found. One of these mutations is apparently purely structural, as it produces a small subunit of phenylalanyl-tRNA synthetase with a reduced molecular weight. This protein is less stable than the wild-type enzyme. These mutations represent useful tools to investigate how the phenylalanyl-tRNA synthetase operon is regulated.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Escherichia coli phenylalanyl-tRNA synthetase operon: transcription studies of wild-type and mutated operons on multicopy plasmids.

The construction of three lambda bacteriophages containing parts of the structural gene for threonyl-tRNA synthetase, thrS, and those for the two subunits of phenylalanyl-tRNA synthetases, pheS and pheT, is described. These phages were used as hybridization probes to measure the in vivo levels of mRNA specific to these three genes. Plasmid pB1 carries the three genes thrS, pheS, and pheT, and strains carrying the plasmid show enhanced levels of mRNA corresponding to these genes. Although the steady-state levels of threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase produced by the presence of the plasmid differed by a factor of 10, their pulse-labeled mRNA levels were about the same. Mutant derivatives of pB1 were also analyzed. Firstly, a cis-acting insertion located before the structural genes for phenylalanyl-tRNA synthetase caused a major decrease in both pheS and pheT mRNA. Secondly, mutations affecting either structural gene pheS or pheT caused a reduction in the mRNA levels for both pheS and pheT. This observation suggests that autoregulation plays a role in the expression of phenylalanyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Physical localisation and cloning of the structural gene for E. coli initiation factor IF3 from a group of genes concerned with translation.

The structural genes for translational initiation factor IF3, threonyl-tRNA synthetase (TRS), the two subunits of phenylalanyl-tRNA synthetase (PRS), and a 12 000 mol. wt. protein of unidentified function are carried by the lambda p2 transducing phage. The localization of these genes on a restriction map of the Escherichia coli DNA insert was achieved by deletion mapping. In addition a set of plasmids carrying fragments of the original phage was constructed and helped to confirm these assignments. One plasmid, containing a 3.3 kb PstI fragment inserted into pBR322, does not code for any of the synthetase genes but causes strains carrying it to overproduce IF3.

Bacteriophage lambda↗
PubMed1979

Genetic organization of the E. coli chromosome around the structural gene for initiation factor IF3 (infC).

A set of lambda transducing phages carrying varying lengths of the E. coli chromosome around the structural gene for initiation factor IF3 (infC) was derived from lambda p2 which is known to carry, besides infC, the structural genes for the alpha subunit of phenylalanyl-tRNA synthetase (pheS), the beta subunit of phenylalanyl-tRNA synthetase (pheT) and the structural gene for threonyl-tRNA synthetase (thrS). The E. coli coding content of these derived phages was analysed by genetic complementation of a set of mutants and by SDS-polyacrylamide gel analysis of the proteins synthesized in UV irradiated cells infected with these phages. The segregation pattern of the different genes among these derived phages indicates that the order of the genes is pheT - pheS - "P12" - (infC, thrS) where infC is probably between "P12" and thrS. "P12" is the structural gene of a 12,000 molecular weight unidentified protein.

Coliphages↗
PubMed1977

A specialized transducing lambda phage carrying the Escherichia coli genes for phenylalanyl-tRNA synthetase.

A lambda phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the alpha and beta subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperature-sensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the alpha and beta subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes. The transducing lambda phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the beta and alpha subunits of PRS, respectively. A third protein with a molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (Mr 78,000) is still unidentified.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Characterization of an E. coli mutant with a thermolabile initiation factor IF3 activity.

A thermosensitive E. coli mutant is described which has at least two defects in vitro: a thermolabile initiation factor IF3 activity and a modified L-phenylalanine: tRNAPhe ligase (EC 6.1.1.20) activity. These two defects cotransduce and are located near 38 min on the new E. coli map. Thermoresistant revertants showing in vitro reversion for one defect also revert in vitro for the other defect. The thermosensitive mutation is recessive to its wild type allele, and in vitro analysis of wild type/ mutant heterodiploïds also show reversion for both defects.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Specialized transducing phage for the initiation factor 3 gene in Escherichia coli.

A previously isolated thermosensitive mutant [Springer, M., Graffe, M. & Grunberg-Manago, M. (6977) Mol. Gen. Genet. 151, 17-26] exhibits two defects in vitro, one in the initiation factor IF3 and the other in the L-phenylalanine: tRNA-Phe ligase (EC 6.1.1.20). Specialized lambda transducing phages that transduced this mutant to thermoresistance were selected. In vitro studies showed that the transductants had a normal IF3 activity. One of these transducing phages was shown to code for a protein synthesized under the control of Escherichia coli promoters, which has the same molecular weight as IF3. This protein crossreacts specifically with IF3 antisera and comigrates with pure IF3 in a two-dimensional gel system.

Bacterial Proteins↗