Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.     

The Escherichia coli threonyl-tRNA synthetase gene contains a split ribosomal binding site interrupted by a hairpin structure that is essential for autoregulation

The expression of the gene encoding Escherichia coli threonyl-tRNA synthetase (ThrRS) is negatively autoregulated at the translational level. ThrRS binds to its own mRNA leader, which consists of four structural and functional domains: the Shine–Dalgarno (SD) sequence and the initiation codon region (domain 1); two upstream hairpins (domains 2 and 4) connected by a single-stranded region (domain 3). Using a combination of in vivo and in vitro approaches, we show here that the ribosome binds to thrS mRNA at two non-contiguous sites: region −12 to +16 comprising the SD sequence and the AUG codon and, unexpectedly, an upstream single-stranded sequence in domain 3. These two regions are brought into close proximity by a 38-nucleotide-long hairpin structure (domain 2). This domain, although adjacent to the 5' edge of the SD sequence, does not inhibit ribosome binding as long as the single-stranded region of domain 3 is present. A stretch of unpaired nucleotides in domain 3, but not a specific sequence, is required for efficient translation. As the repressor and the ribosome bind to interspersed domains, the competition between ThrRS and ribosome for thrS mRNA binding can be explained by steric hindrance.     

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.     

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.     

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.     

Molecular mimicry in translational control of E. coli threonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules.

We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively. The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex. Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly. As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site. The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity. It further shows that the tRNA-like operator obeys to tRNA identity rules.     

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.     

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site.

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.     

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.     

Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.     

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.     

Threonyl-Transfer Ribonucleic Acid Synthetase from Escherichia coli: Subunit Structure and Genetic Analysis of the Structural Gene by Means of a Mutated Enzyme and of a Specialized Transducing Lambda Bacteriophage

Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.     

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.     

Escherichia coli Mutants Overproducing Phenylalanyl- and Threonyl-tRNA Synthetase

The structural genes for threonyl-tRNA synthetase (ThrRS) and phenylalanyl-tRNA synthetase (PheRS) are closely linked on the Escherichia coli chromosome. To study whether these enzymes share a common regulatory element, we have investigated their synthesis in mutants which were selected for overproduction of either ThrRS or PheRS. It was found that mutants isolated previously for overproduction of ThrRS as strains resistant to the antibiotic borrelidin (strains Bor Res 3 and Bor Res 15) did not show an elevated level of PheRS. PheRS-overproducing strains were then isolated as revertants of strains with structurally altered enzymes. Strain S1 is a temperature-resistant derivative of a temperature-sensitive PheRS mutant, and strain G118 is a prototrophic derivative of a PheRS mutant which shows phenylalanine auxotrophy as a consequence of an altered K(m) of this enzyme for the amino acid. In both kinds of revertants, S1 and G118, the concentration of PheRS and ThrRS was increased by factors of about 2.5 and 1.8, respectively, whereas the level of other aminoacyl-tRNA synthetases was not affected by the mutations. Genetic studies showed that the simultaneous overproduction of PheRS and ThrRS in revertants G118 and S1 is based upon gene amplification, since this property was easily lost after growing the cells in the absence of the selective stimulus, and since this loss could be prevented by the presence of the recA allele. By similar criteria, the four- and eightfold overproduction of ThrRS in strains Bor Res 3 and Bor Res 15, respectively, was very stable genetically, indicating that it is caused by a mutational event other than gene amplification. From these results, we conclude that the concomitant increase of PheRS and ThrRS in strains G118 and S1 is an expression of gene duplication and not of a joint regulation of these two aminoacyl-tRNA synthetases. This conclusion is further supported by the result that, in mutant G118 as well as in its parental strain G1, growth in minimal medium lacking phenylalanine led to an additional twofold increase of their PheRS concentration. This increase was restricted to the PheRS, since the level of other aminoacyl-tRNA synthetases, including the ThrRS, stayed unchanged.     

Regulation of HSP70 synthesis by messenger RNA degradation.

When Drosophila cells are heat shocked, hsp70 messenger RNA (mRNA) is stable and is translated at high efficiencies. During recovery from heat shock, hsp70 synthesis is repressed and its messenger RNA (mRNA) is degraded in a highly regulated fashion. Dramatic differences in the timing of repression and degradation are observed after heat treatments of different severities. The 3' untranslated region (UTR) of the hsp70 mRNA was sufficient to transfer this regulated degradation to heterologous mRNAs. Altering the translational efficiency of the message or changing its natural translation-termination site did not alter its pattern of regulation, although in some cases it changed the absolute rate of degradation. We have previously shown that hsp70 mRNA is very unstable when it is expressed at normal growth temperatures (from a metallothionein promoter). We report here that the 3' untranslated region of the hsp70 mRNA is responsible for this instability as well. We postulate that a mechanism for degrading hsp70 mRNA pre-exists in Drosophila cells, that it is inactivated by heat shock and that it is the reactivation of this mechanism that is responsible for hsp70 repression during recovery. This degradation system may be the same as that used by other unstable mRNAs.     

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA^AA formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.