FIS-dependent trans-activation of tRNA and rRNA operons of Escherichia coli.

Two mechanisms controlling stable RNA synthesis have been described: growth rate-dependent control and stringent response. Although the mechanism underlying growth rate-dependent control is still a matter of dispute, this control is commonly assumed to operate through repression of transcription initiation of stable RNA operons. The same is true for the stringent response. Here we show that the cell utilizes an additional control system operating through activation of the thrU(tufB) operon. This operon, the tyrT and the rrnB operon share a common trans-activating protein that binds to cis-acting DNA regions upstream of the promoters of the two tRNA operons and of the P1 promoter of the rrnB operon. Conceivably, more stable RNA operons may be regulated by trans-activation. Both in vivo and in vitro experiments show that the Escherichia coli protein FIS (Factor for Inversion Stimulation) is involved in the trans-activation. This protein is known to stimulate the inversion of various DNA segments by binding to cis-acting recombinational enhancers and functions as a host factor for the bacteriophages Mu and Lambda.

Cloning and sequencing of the tuf genes of Streptomyces coelicolor A3(2).

Two tuf genes are present in Streptomyces coelicolor A3(2), which have been cloned and sequenced. These genes show a high degree of nucleotide sequence identity to the tuf1 and tuf3 genes of Streptomyces ramocissimus: the tuf1 genes are 94% identical, the tuf3 genes 87%. S. coelicolor tuf1 encodes a protein of 396 amino acids, while tuf3 encodes a protein of 391 amino acids.

Cloning and sequence analysis of the gene encoding Methylophilus methylotrophus cytochrome c", a unique protein with a perpendicular orientation of the histidinyl ligands.

Cytochrome c" from Methylophilus methylotrophus is an unusual monohaem protein that undergoes a major redox-linked spin-state transition: one of the two axial histidines bound to the iron in the oxidised form is detached upon reduction and a proton is taken up. A 3.5-kb DNA fragment, containing the gene encoding cytochrome c" (cycA), has been cloned and sequenced. The cytochrome c" gene codes for a pre-protein with a typical prokaryotic 20-residue signal sequence, suggesting that the protein is synthesised as a precursor which is processed during its secretion into the periplasm. The C-terminus of cytochrome c" has homology with the corresponding region of an oxygen-binding haem protein (SHP) from phototrophically grown Rhodobacter sphaeroides. SHP is similar in size and in the location of its haem-binding site. Immediately downstream from cytochrome c" a second open reading frame (ORF) codes for a 23-kDa protein with similarity to the cytochrome b-type subunit of Ni-Fe hydrogenase. The possibility of coordinated expression of cycA and this ORF is discussed.

Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.

1H NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase.

The 600 MHz 1H NMR spectrum of tyrosinase (31 kDa) of Streptomyces antibioticus in the oxidized, chloride-bound form is reported. The downfield part of the spectrum (15-55 ppm) exhibits a large number of paramagnetically shifted signals. The paramagnetism is ascribed to a thermally populated triplet state. The signals derive from six histidines binding to the metals through their Nepsilon atoms. There is no evidence for endogenous bridges. The exchange coupling, -2J, amounts to 298 cm(-1). In the absence of chloride the peaks broaden. This is ascribed to a slowing down of the electronic relaxation. The exchange coupling decreases to -2J=103 cm(-1).

EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus.

The [Cu(I)-Cu(II)] half-met form of the dinuclear copper site of tyrosinase has been probed by continuous wave electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy in the presence and absence of inhibitors. In all cases the EPR spectrum is indicative of a d(x(2)-y(2)) ground state for the unpaired electron. From the cross-peaks observed in the HYSCORE spectra, proton hyperfine coupling constants were obtained that are compatible with a hydroxide ion in an equatorial coordination position of the paramagnetic copper. After changing the water solvent to D(2)O or after addition of the inhibitors p-nitrophenol or L-mimosine, the proton signals disappear. The relevance of these findings for understanding the catalytic cycle is discussed.

Introduction of a CuA site into the blue copper protein amicyanin from Thiobacillus versutus.

The C-terminal loop of the blue copper protein amicyanin, which contains three of the four active site ligands, has been replaced with a CuA binding loop. The purple protein produced has visible and EPR spectra identical to those of a CuA centre. Recent evidence strongly suggests that the CuA centre of cytochrome c oxidase and the A centre of nitrous oxide reductase are similar and are both binuclear. It therefore follows that the purple amicyanin mutant created here also possesses a binuclear CuA centre.

Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell.

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.

Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli.

Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli. Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu. A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting. The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately. These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.

The elongation factor EF-Tu from E. coli binds to the upstream activator region of the tRNA-tufB operon.

The polypeptide chain elongation factor EF-Tu of Escherichia coli is encoded by two genes, tufA and tufB, located in two different operons. Experiments in which either tufA or tufB was inactivated demonstrated that expression of the tRNA-tufB operon is dependent on a functioning tufA and thus on EF-Tu (1, to be published). In order to study a possible role of EF-Tu as trans-activator of the tRNA-tufB operon, we have investigated in vitro binding of an EF-Tu. GDP preparation to various DNA fragments of the operon. We demonstrate that specific binding occurs to a cis-acting region delimited from position -134 to the promoter, previously shown to enhance tufB transcription. Electrophoretic retardation assays reveal the formation of maximally three protein/DNA complexes, indicating that more than one protein molecule can bind to the DNA. The EF-Tu preparation used was obtained by affinity chromatography and appeared to be 95% pure. It lost its DNA binding activity upon further purification. That EF-Tu is nonetheless involved in the DNA binding is suggested by the observation that none of the three complexes is formed in the presence of kirromycin, an antibiotic that binds EF-Tu with high specificity. If so, EF-Tu.GDP most likely binds to the activator region of the tRNA-tufB operon in combination with another non-identified protein or component.

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors.

Read-through of nonsense codons has been studied in wild-type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF-Tu. The two phenomena differ essentially. Readthrough of UGA in wild-type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid-borne EF-Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF-Tu. Strains of E. coli harbouring mutant species of the elongation factor EF-Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF-Tu species: EF-TuAR(Ala 375----Thr) and EF-TuBo(Gly 222----Asp). Cells harbouring EF-TuAR(Ala 375----Thr) and wild-type EF-TuB, or wild-type EF-TuA and EF-TuBo(Gly 222----Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF-Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF-Tu molecules on the ribosome.

A comparative study of the ribosomal RNA operons of Streptomyces coelicolor A3(2) and sequence analysis of rrnA.

S. coelicolor A3(2) contains six ribosomal RNA operons. Here we describe the cloning of rrnA, rrnC and rrnE, thereby completing the cloning of all operons. Southern hybridisation of genomic DNA with a heterologous probe from the E.coli rrnB 16S rRNA gene showed differences in hybridisation among the six rRNA operon-containing bands. The nucleotide sequence of the 16S rRNA gene and the upstream region of rrnA was determined and compared with the corresponding sequence of rrnD, showing that the 16S rRNA genes are 99% identical. Substantial differences were found, however, in the upstream regions corresponding to the P1 and P2 promoters of rrnD. Southern analysis showed that some of the other rRNA operons of S.coelicolor A3(2) also differed in this part of the upstream region.

The role of FIS in trans activation of stable RNA operons of E. coli.

The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.

Isolation and functional analysis of histidine-tagged elongation factor Tu.

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.

FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is independent of nutritional factors. The well-known growth rate-dependent control, displayed by exponentially growing cells studied under various nutritional conditions, is governed by two regulatory mechanisms: repression, presumably by ribosome feedback inhibition, and stimulation by trans activation. FIS allows very fast bacterial growth.

Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae.

The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteriods compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two- to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown, negative regulation mechanism.

The role of EF-Tu in the expression of tufA and tufB genes.

We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu in Escherichia coli. To this aim we have determined the intracellular concentrations of EF-TuA and EF-TuB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild-type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady-state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF-TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF-TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single-site mutation of tufA, however, rendering EF-TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression of tufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild-type cells the EF-Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF-Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA-tufB transcriptional unit as a template. They show that addition to this system of EF-Tu in concentrations 2-5% of the endogenous amount results in strong inhibition of EF-Tu synthesis. We hypothesize that EF-Tu acts as an autogenous repressor, inhibiting tufB expression post-transcriptionally.

tuf gene dosage effects on the intracellular concentration of EF-TuB.

In this paper we have studied the effect of raising the intracellular EF-Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF-Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (PL) of phage lambda. Transcription from PL can be repressed at low temperature by a temperature-sensitive repressor and activated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of PL. Cells carrying either plasmid were shown to contain an almost doubled amount of EF-Tu at temperatures from 28 degrees C to 37 degrees C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF-Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37 degrees C to 43 degrees C. The multicopy plasmid pTuB1 described by Miyajima et al. [FEBS Lett. 102, 207-210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid-borne tufB. Transformation with either plasmid raised the intracellular EF-Tu concentration by 30-60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF-Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF-Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.

The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control.

In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5'-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in sigma hrdB, the principal sigma factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNA(Leu) for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.