Autogenous repression of Escherichia coli threonyl-tRNA synthetase expression in vitro

Escherichia coli threonyl-tRNA synthetase (EC 6.1.1.3) expression has been examined in an acellular protein-synthesizing system programmed with a plasmid DNA carrying thrS, infC, pheS, and pheT, the gene for threonyl-tRNA synthetase, initiation factor 3, and the two protomers of phenylalanyl-tRNA synthetase (EC 6.1.1.20), respectively. The initial rate of synthesis of L-[35S]methionine-labeled threonyl-tRNA synthetase is markedly reduced by the addition of homogeneous RNase-free threonyl-tRNA synthetase to the assay, not by that of phenylanyl- or tyrosyl-tRNA synthetase (EC 6.1.1.1). The inhibition is 50% in the presence of 0.25 microM threonyl-tRNA synthetase and reaches 90% with 2 microM enzyme. Synthesis of mRNA in the acellular DNA-dependent protein-synthesizing system has been measured by molecular hybridization to gene-specific lambda DNA probes corresponding to thrS, pheS, and pheT. The addition to the assay of 2 microM threonyl-tRNA synthetase does not affect the extent of mRNA hybridizing to the thrS-specific DNA probe. This result is interpreted as reflecting an effect of the synthetase on its expression at the translational level. Analysis of the DNA sequence of the thrS gene predicts several potential secondary structures capable of forming in the thrS mRNA. One of these potential structures is a cloverleaf. The possible role of such structures in controlling expression of thrS is discussed.     

Threonyl-transfer ribonucleic acid synthetase and the regulation of the threonine operon in Escherichia coli.

Two threonine-requiring mutants with derepressed expression of the threonine operon were isolated from an Escherichia coli K-12 strain containing two copies of the thr operon. One of them carries a leaky mutation in ilvA (the structural gene for threonine deaminase), which creates an isoleucine limitation and therefore derepression of the thr operon. In the second mutant, the enzymes of the thr operon were not repressed by threonine plus isoleucine; the threonyl-transfer ribonucleic acid(tRNA) synthetase from this mutant shows an apparent Km for threonine 200-fold higher than that of the parental strain. The gene, called thrS, coding for threonyl-tRNA synthetase was located around 30 min on the E. coli map. The regulatory properties of this mutant imply the involvement of charged threonyl-tRNA or threonyl-tRNA synthetase in the regulation of the thr operon.     

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.     

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.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.     

Regulation of argA operon expression in Escherichia coli K-12: cell-free synthesis of beta-galactosidase under argA control.

Regulation of argA operon expression in Escherichia coli K-12 was studied in a cell-free, deoxyribonucleic acid-dependent, enzyme-synthesizing system. lambdaAZ-7 deoxyribonucleic acid, which carries a fusion of the lacZ structural gene to the argA operon so that beta-galactosidase synthesis is under argA regulation, was used as the template. To eliminate extraneous readthrough from lambda promoters, lambda repressor was introduced into the synthesis mixtures by preparing the S-30 component from a strain (514X5a-12-29) that carries a multicopy hybrid plasmid (pKB252) containing the lambdacI gene. Under these conditions beta-galactosidase synthesis was repressed 90% by the arginine repressor when a sufficient concentration of L-arginine was present. This repression could be overcome by escape synthesis when the lambdaAZ-7 deoxyribonucleic acid concentration in the synthesis mixtures was increased. Guanosine 3'-diphosphate-5'-diphosphate stimulated beta-galactosidase synthesis from this template.     

Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.     

The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene.

Expression of thrS, the gene encoding Escherichia coli threonyl-tRNA synthetase, is negatively autoregulated at the translational level. Regulation is due to the binding of threonyl-tRNA synthetase to its own mRNA at a site called the operator, located immediately upstream of the initiation codon. The present work investigates the relationship between regulation and mRNA degradation. We show that two regulatory mutations, which increase thrS expression, cause an increase in the steady-state mRNA concentration. Unexpectedly, however, the half-life of thrS mRNA in the derepressed mutants is equal to that of the wild-type, indicating that mRNA stability is independent of the repression level. All our results can be explained if one assumes that thrS mRNA is either fully translated or immediately degraded. The immediately degraded RNAs are never detected due to their extremely short half-lives, while the fully translated messengers share the same half-lives, irrespective of the mutations. The increase in the steady-state level of thrS mRNA in the derepressed mutants is simply explained by an increase in the population of translated molecules, i.e. those never bound by the repressor, ThrRS. Despite this peculiarity, thrS mRNA degradation seems to follow the classical degradation pathway. Its stability is increased in a strain defective for RNase E, indicating that an endonucleolytic cleavage by this enzyme is the rate-limiting process in degradation. We also observe an accumulation of small fragments corresponding to the 5' end of the message in a strain defective for polynucleotide phosphorylase, indicating that, following the endonucleolytic cleavages, fragments are normally degraded by 3' to 5' exonucleolytic trimming. Although mRNA degradation was suspected to increase the efficiency of translational control based on several considerations, our results indicate that inhibition of mRNA degradation has no effect on the level of repression by ThrRS.     

Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis.

With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed.     

Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors.

The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.