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L Bosch

Publications and source records attributed to L Bosch.

At least 19 recordsLinked to original sources
PubMed1992

Inactivation of the fis gene leads to reduced growth rate.

The FIS protein (factor for inversion stimulation) is known to activate the transcription of rRNA and tRNA operons in Escherichia coli. In this report the growth behaviour of a fis bacterium was studied under steady-state conditions and in competition experiments with a wild-type bacterium in a chemostat. The growth rate of the fis bacterium was reduced as compared to that of an isogenic wild-type and this reduction increased linearly with increasing growth rate. A similar result was obtained in competition experiments in a chemostat, with the selection factor increasing linearly with the growth rate.

Carrier Proteins↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

The mechanism of trans-activation of the Escherichia coli operon thrU(tufB) by the protein FIS. A model.

Transcription of the thrU(tufB) operon is trans-activated by the protein FIS which binds to the promoter upstream activator sequence (UAS). Deletions of parts of the UAS and insertions show that optimal trans-activation requires occupation by FIS of the two FIS-binding regions on the UAS and specific helical positioning of these regions. On the basis of these and other data, a model for the mechanism of thrU(tufB) trans-activation by FIS is proposed. This model implies that the mechanisms underlying stimulation by FIS of two totally different processes: inversion of viral DNA segments and transcription of stable RNA operons, are essentially the same.

Bacterial Proteins↗
PubMed1992

Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability.

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1992

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.

Carrier Proteins↗
PubMed1991

Thermal stability of turnip yellow mosaic virus RNA: effect of pH and multivalent cations on RNA deaggregation and degradation.

Light scattering studies of RNA isolated from turnip yellow mosaic virus (TYMV) revealed a molar mass of 1.9.10(6) g mol-1, which is close to the value of 2.0.10(6) g mol-1 published for intact genomic TYMV RNA (2M RNA). However, gel electrophoresis under denaturing conditions demonstrated that only 30-40% of this native RNA was 2M RNA. Sucrose gradient centrifugation revealed the occurrence of a series of smaller RNA size classes, the mass ratios of which were greatly influenced by the pH of the solution and the presence of EDTA. These results suggest that native TYMV RNA preparations originally contain a mixture of intact RNA particles and of aggregates of RNA fragments with the same molar mass of about 2.10(6) g mol-1, and that the size classes are intermediates in the deaggregation process of the degraded genomic TYMV RNA. The native RNA displayed pH-dependent deaggregation and degradation. The degradation process of 2M RNA followed (pseudo) first-order kinetics. Lower degradation rates were observed for RNA depleted of divalent cations and polyamines. For depleted 2M RNA an enthalpy of activation of about 100 kJ mol-1 and an almost zero entropy of activation was calculated. Similar values were also found for depleted E. coli ribosomal RNAs and depleted MS2 RNA, demonstrating that all RNAs are equally vulnerable to degradation. In the presence of multivalent cations the activation enthalpy for 2M TYMV RNA degradation increased to 150 kJ mol-1 and the entropy of activation to 150 J K-1 mol-1, indicative for a different degradation mechanism.

Cations, Divalent↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Electrophoresis, Agar Gel↗
PubMed1991

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA.

Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

FIS-induced bending of a region upstream of the promoter activates transcription of the E coli thrU(tufB) operon.

The upstream activator sequence (UAS) of the thrU(tufB) operon, which is the target of the trans-activating protein FIS, has a bent structure. Here we show that the center of bending lies around position -95, between the two FIS-binding regions. Studies with fis+ and fis- cells show that FIS-induced bending of the UAS plays a major role in the trans-activation of the thrU(tufB) operon. This has been concluded from the finding that insertions of small DNA segments, comprising less than one or two complete helix turns, in the junction of the UAS and the RNA polymerase-binding site reduce transcription significantly. Partial restoration of transcriptional activity occurs when one or more full helix turns are inserted. These data are in line with but do not prove that a direct interaction between FIS and RNA polymerase is involved in trans-activation. A role of bending per se resulting from FIS/DNA interaction cannot be excluded.

Base Sequence↗
PubMed1990

Aminoacylation of 3' terminal tRNA-like fragments of turnip yellow mosaic virus RNA: the influence of 5' nonviral sequences.

The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides. A previous kinetic study on 3' terminal TYMV RNA fragments that contain the tRNA-like structure and a 5' nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al. (1988) Biochimie 70, 1719-1727). Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5' end of TYMV RNA transcripts was reduced. The influence of these 5' proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments. A structure that deviates from the tRNA-like structure was found in some of the transcripts. The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain. Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.

Base Sequence↗
PubMed1990

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.

Bacteriophage mu↗
PubMed1990

Potential binding sites of the trans-activator FIS are present upstream of all rRNA operons and of many but not all tRNA operons.

FIS, the Escherichia coli protein that stimulates the inversion of various DNA segments by binding to a recombinational enhancer, trans-activates a number of stable RNA operons and binds to the upstream activator sequence (UAS) of these operons (Nilsson et al. (1990) EMBO J. 9, 727). In a search for potential FIS-binding sites we have compared UASs of other stable RNA operons with a consensus FIS-binding sequence, compiled by comparing recombinational enhancers. Such sites can thus be recognized upstream of all rRNA and 13 tRNA operons. Matching with the consensus sequence varied, suggesting that the affinity of FIS for the sites differed. Accordingly, FIS binding to an upstream sequence of the metY(nusA) operon was found to be weaker than that to the UAS of the thrU(tufB) operon. No FIS binding sites were found upstream three tRNA operons.

Anticodon↗
PubMed1990

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.

Carrier Proteins↗
PubMed1990

RNA pseudoknots: translational frameshifting and readthrough on viral RNAs.

Ribosomal frameshifting on retroviral RNAs has been proposed to be mediated by slippage of two adjacent tRNAs into the -1 direction at a specific heptanucleotide sequence. Here we report a computer-aided analysis of the structure around the established or putative frameshift sites in a number of retroviral, coronaviral, toroviral, and luteoviral RNAs and two dsRNA yeast viruses. In almost all cases a stable hairpin was predicted four to nine nucleotides downstream of the shifty heptanucleotide. More than half of the resulting hairpin loops give rise to potential pseudoknotting with sequences downstream of this hairpin. Especially in the case of the shifty heptanucleotides U UUA AAC and G GGA AAC, stable downstream pseudoknots are present. Indications were also found for the presence of pseudoknots downstream of amber stop condons at readthrough sites in some retroviral RNAs.

Base Sequence↗
PubMed1989

Biochemical and biophysical analysis of pseudoknot-containing RNA fragments. Melting studies and NMR spectroscopy.

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site-directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion-exchange HPLC, yielded a pure preparation of a 3'-terminal, 112-nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T7 RNA polymerase, resulted in the isolation of an 88-nucleotide fragment. Finally, a 44-nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy. Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44-nucleotide fragment. Also the analogue of the T-stem and T-loop of the tRNA-like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+-containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species. To obtain direct biophysical evidence for base-pairing and stacking interactions in the pseudoknot, NMR studies were initiated. The first proton-NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112-nucleotide and 88-nucleotide fragment are too complicated to be solved at present. In the case of the 44-nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.

Base Sequence↗
PubMed1989

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.

Bacterial Proteins↗