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M Ehrenberg

Publications and source records attributed to M Ehrenberg.

At least 73 records · Page 4Linked to original sources
PubMed1991

How does ppGpp affect translational accuracy in the stringent response?

With an in vitro poly(Phe) synthesis system we have tested recent models concerning translational accuracy in the stringent response during aminoacid starvation. We have found that cognate, deacylated tRNA of very high concentrations is unable to block the A-site. No influence of EF-Tu.ppGpp on ribosomal proofreading has been found. Alternative mechanisms to keep translational errors low by the stringent response are discussed.

Amino Acids↗
PubMed1991

[HCV antibodies in selected patient groups and blood donors].

In a multicenter study in three eastern German blood banks, we searched for HCV antibodies in polytransfused patients, unpaid donors, hemodialysis patients and hemophiliacs. The high seropositivity of HCV antibodies in polytransfused patients shows the danger of the transmission of HCV through the transfusion of blood. Therefore, blood banks should consider making HCV antibody screening of the blood of donors a part of the policy to reduce NANB PTH.

Blood Banks↗
PubMed1990

Impaired in vitro kinetics of EF-Tu mutant Aa.

The kirromycin-resistant EF-Tu mutant Aa, previously shown to be an antisuppressor for nonsense and missense suppressor tRNAs, has been characterised in a poly(U)-primed translation system in vitro. Two major defects were found in the function of the mutant. First, the dissociation constant for Aa binding to Phe-tRNA(Phe) was increased tenfold compared to wild-type EF-Tu. Second, kcat/Km for the interaction between the EF-Tu.GTP.aa-tRNA complex and the ribosome was decreased by the mutation to one third of its wild-type value. No differences were observed between mutant and wild-type factor in the regeneration of EF-Tu.GTP from EF-Tu.GDP via EF-Ts or in the mistranslation frequency by Leu-tRNA(4Leu). The relation between the in vitro results and the mutant phenotype in vivo is discussed.

Bacterial Proteins↗
PubMed1990

[Progress in the development of a detection test for parenteral non-A, non-B hepatitis--results of enzyme immunoassay for anti-hepatitis C virus].

After more than one decennium of international research work the doubtless identification of the causative agents of the non A-non B-hepatitis (NANBH) has not yet been successful. 1988, however, a viral genome of the parenteral NANBH could be isolated, on which basis an EIA was built up. By means of this anti-HCV-ELISA altogether 413 sera were tested. In 262 sera of 154 women of a NANBH-group with homogeneous source of infection (contaminated anti-D-immunoglobulin) in 74% positive reactions were the result. This and the extensive reproducibility of the test results in identical patients speak for the fact that the recombinant antigen underlying the test really belongs to the parenteral NANBH-group. In the group of the sporadic, however, only in one case a positive reaction was achieved, which supports the thesis of at least two parenteral causative agents of NANBH. The deep-freezing storage of patients' sera lasting up to 8 years did not lead to the failure of the test. The reasons for non-reactive tests were discussed.

Adult↗
PubMed1990

How many EF-Tu molecules participate in aminoacyl-tRNA binding and peptide bond formation in Escherichia coli translation?

We have observed that two EF-Tu.GTP cycles are required to make one peptide bond during steady-state translation in an accurate and fast poly(U) translation system prepared from Escherichia coli. We have also found that there are two complexes of EF-Tu.GTP bound to one molecule of aminoacyl-tRNA under our experimental conditions. We suggest, on the basis of these data, that aminoacyl-tRNA enters the ribosomal A-site in a pentameric complex together with two EF-Tu and two GTP molecules. When the tRNA is delivered to the ribosome two GTP molecules are hydrolyzed. It is possible that the functional role of such an EF-Tu dimer is related to the function of the two L7/L12 dimers in the large ribosomal subunit.

Escherichia coli↗
PubMed1990

Ribosomal RNA and protein mutants resistant to spectinomycin.

We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild-type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF-G) cycle supported by wild-type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF-G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.

Escherichia coli↗
PubMed1988

Is translation inhibited by noncognate ternary complexes?

We studied the influence of an error-prone isoacceptor (tRNALeu4), as well as an intermediate (tRNALeu2) and a weak (tRNAVal) competitor of tRNAPhe on the poly(Phe) synthesis rate. Even at very high excess concentrations of these noncognate ternary complexes there was no significant effect on the translation rate. Our result argues against the assertion that in vivo translation is slowed down by noncognate tRNA and favours the hypothesis that the incorrect ternary complex concentrations are too low to saturate the ribosomes in vivo.

Binding, Competitive↗
PubMed1988

Mutations in ribosomal proteins L7/L12 perturb EF-G and EF-Tu functions.

In vitro cycling rates of E. coli ribosomes and of elongation factors EF-Tu and EF-G have been obtained and these are compatible with translation rates in vivo. We show that the rate of translocation is faster than 50 s-1 and therefore that the EF-G function is not a rate limiting step in protein synthesis. The in vivo phenotype of some L7/L12 mutants could be accounted for by perturbed EF-Tu as well as EF-G functions. The S12 mutants that we studied were, in contrast, only perturbed in their EF-Tu function, while their EF-G interaction was not impaired in relation to wild type ribosomes.

Bacterial Proteins↗
PubMed1986

How do combinations of rpsL- and miaA- generate streptomycin dependence?

Petrullo et al. (1983) have studied the consequences of combining a mutation (rpsL-) that normally generates streptomycin resistant (Smr) ribosomes with a mutation (miaA-) that leads to loss of a tRNA hypermodification. They found surprisingly that such doubly mutant bacteria become streptomycin dependent (Smd). Here, we show in vitro that ribosomes purified from an Smr mutant behave very like Smd ribosomes when they are combined with tRNA from an miaA- mutant. Our analysis suggests that proofreading becomes excessively intense when the mutant components are combined, and that this reduces the efficiency of translation to the very low levels characteristic of Smd ribosomes. We show that Sm increases the efficiency of translation in vitro by suppressing the proofreading flows. We suggest that this will explain the growth stimulatory effect of Sm on the rpsL-, miaA- double mutants.

Escherichia coli↗
PubMed1986

Counting cycles of EF-Tu to measure proofreading in translation.

A new method (T. Ruusala et al., 1982, EMBO J. 1, 75-78, 741-748) for analyzing kinetic proofreading in translation is described. An in vitro system is arranged so that its rate of polypeptide synthesis is determined by the release rate of GDP from EF-Tu in the absence of EF-Ts. This enables the counting of the number of EF-Tu cycles for correct as well as for incorrect peptide bonds. The necessary equations are derived and the approximations involved in these are discussed together with data from experiments not previously described.

Guanosine Diphosphate↗
PubMed1984

Hyper-accurate ribosomes inhibit growth.

We have compared both in vivo and in vitro translation by ribosomes from wild-type bacteria with those from streptomycin-resistant (SmR), streptomycin-dependent (SmD) and streptomycin-pseudo-dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild-type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild-type in the absence of Sm. The antibiotic stimulates both the growth rate and the translation rate of SmP bacteria by approximately 2-fold, but it simultaneously increases the nonsense suppression rate quite dramatically. Kinetic experiments in vitro show that the greater accuracy and slower translation rates of mutant ribosomes compared with wild-type ribosomes are associated with much more rigorous proofreading activities of SmR, SmD and SmP ribosomes. Sm reduces the proofreading flows of the mutant ribosomes and stimulates their elongation rates. The data suggest that these excessively accurate ribosomes are kinetically less efficient than wild-type ribosomes, and that this inhibits mutant growth rates. The stimulation of the growth of the mutants by Sm results from the enhanced translational efficiency due to the loss of proofreading, which more than offsets the loss of accuracy caused by the antibiotic.

Drug Resistance, Microbial↗
PubMed1984

ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis.

The inhibition of elongation factors G, Tu and Ts by ppGpp was studied in vitro in a translation system with missense frequency and elongation rate similar to those in vivo. ppGpp inhibits EF-G with KI = 6 X 10(-5) M. When ppGpp is in twofold excess over GTP and EF-G is the rate-limiting component, the elongation rate is reduced twofold by ppGpp. EF-Tu is inhibited with KI = 7 X 10(-7) M in the absence of EF-Ts. When EF-Ts is added, the binding of ppGpp to EF-Tu becomes successively weaker. 1/KI depends linearly on 1/[Ts] and the intercept at the abscissa gives KI = 4 X 10(-5) M. This reflects the binding of ppGpp to the binary TuTs complex. The slope reveals that the binding of EF-Ts to the TuMS binary complex is strong (10(-6) M). ppGpp may thus inhibit the cycling of EF-Tu indirectly by the removal of the free EF-Ts by its adsorption to TuMS, as well as directly by simple binding to Tu. EF-Tu inhibition by ppGpp can be fully reversed by high levels of aminoacyl-tRNA only in the presence of EF-Ts and at low ribosomal activity. Our in vitro observations have been extrapolated to in vivo conditions with conclusions as follows: Under strong amino acid starvation ppGpp in twofold excess over GTP cannot reduce significantly the elongation rate of ribosomes and thereby restore the errors to their normal levels as in the stringent response. Under weak starvation, in contrast, a significant rate reduction can be achieved by the trapping of EF-Ts in complex with TuppGpp.

Bacterial Proteins↗
PubMed1984

Gas-mediated vitreous compression: an experimental alternative to mechanized vitrectomy.

We have developed a simple technique in rabbit and baboon eyes that utilizes intravitreally injected perfluoropropane gas, which expands slowly to efficiently compress and displace nearly completely the vitreous body. There is cataract formation after extended contact of the gas bubble with the lens. However, it is rapidly reversible by reducing the duration of lens contact (gas-fluid exchange) and by using young animals. No long-term alterations in intraocular pressure or retinal function were observed, as determined by electroretinography, during the 4-month test period. Gross examination and scanning electron microscopy revealed that the vitreous cavity, shortly after full expansion of the gas bubble, was practically free of collagen. The vitreous body had been detached from most of the retina and compressed into a collagenous strand between the optic nerve head and lens in the rabbit eyes, and there was also a dense collagen accumulation on the inferior retinal surface and anterior vitreous base in the rabbits and the baboon. Large areas of the retina and medullary wings were stripped of overlying collagen. By the end of 4 months, the compressed vitreous body had expanded to become an irregular structure that remained separated from the retina in areas of previous detachment. Mechanized vitrectomy is a difficult procedure often needed in experimental work. We believe that the vitreous compression and gas-fluid exchange technique is a valid alterative to a mechanical approach. We also believe that we have a model that simulates the human situation of posterior vitreous detachment and vitreous syneresis.

Animals↗