Biomedical subjects

M Ehrenberg

Publications and source records attributed to M Ehrenberg.

At least 19 recordsLinked to original sources

PubMed1996

Rate of translation of natural mRNAs in an optimized in vitro system.

We report results on in vitro translation of an mRNA coding for elongation factor TuB which was in vitro transcribed from the tufB gene from Escherichia coli. Translation occurs at a rate of about 10 codons per second, which is close to the in vivo rate. Protein elongation obeys Michaelis-Menten kinetics with respect to the concentrations of the elongation factors EF-Tu and EF-G in the translation system. The measured K(m) values for EF-Tu and EF-G are 10 and 0.25 microM, respectively. The obtained k(cat) and K(m) values were used to estimate the average k(cat)/K(m) of about 24 x 10(6) s-1 M-1 for the interaction of individual EF-Tu*GTP*aa-tRNA complexes with ribosomes. The estimated k(cat)/K(m) value for EF-G is 36 x 10(6) s-1 M-1. We have also studied translation with a "hyperaccurate" ribosome variant that is pseudodependent on streptomycin (SmP). We have found that SmP ribosomes translate the TuB mRNA significantly slower than wild-type ribosomes do. This is mainly due to a threefold lower k(cat)/K(m) for the interaction of EF-Tu*GTP*aa-tRNA complexes with SmP ribosomes.

Animals↗

PubMed1996

Mutants of EF-Tu defective in binding aminoacyl-tRNA.

Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120 Gln, Gln124 Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu.GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k cat/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120 Gln, Gln124 Arg and Tyr160 Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu.GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124 Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160 Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

Guanosine Triphosphate↗

PubMed1996

Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

Amino Acid Sequence↗

PubMed1996

Hypothesis: hypersensitive plasmid copy number control for ColE1.

Initiation of replication of the plasmid ColE1 is primed by the cis-acting RNA II. Copy numbers are regulated by inhibition of RNA II by the antisense RNA I, whose concentration is proportional to the plasmid concentration. This inhibition is enhanced by a protein. Rom, and takes place during a time set by the transcription of 250 bases of the gene for RNA II. When this transcription is dominated by several steps of about equal duration, the probability for RNA II to prime DNA replication is approximately determined by e-constant[RNA I]. For large values of the "constant" small changes in [RNA I] give large variations in the priming probability. It is shown, first, that this type of mechanism can reduce the rate of plasmid loss and enable single copies of ColE1 to duplicate at a well-defined time in the cell cycle; second, that when the rate of initiation of transcription of RNA II increases, plasmid losses decrease and the distribution of single copy duplication times becomes narrower; third, that the action of Rom may further reduce plasmid losses and further narrow the distribution of duplication times in the single-copy case.

Bacterial Proteins↗

PubMed1995

Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing.

A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis.

Bacteria↗

PubMed1995

A model for copy number control of the plasmid R1.

A new model for copy number control of the plasmid R1 has been developed. It takes into account that initiation of replication of R1 requires a large number of cis-acting proteins (RepA). The theory explains how plasmid production rates respond to shifts in external conditions. It predicts the observed "eclipse" times between two plasmid duplications as well as the replication time for "runaway" plasmids lacking the antisense inhibitor CopA. The model also describes how the use of many cis-acting RepAs can lead to a tight coupling between cell and plasmid cycles that minimizes the rate of the plasmid loss. The results may be used as a guideline for construction of low copy number plasmids with high maintenance stability.

Computer Simulation↗

PubMed1995

Stoichiometry for the elongation factor Tu.aminoacyl-tRNA complex switches with temperature.

In bacterial protein synthesis binding of an aminoacyl-transferRNA (aa-tRNA) to the ribosomal acceptor site (A-site) is catalyzed by elongation factor Tu (EF-Tu). Two guanosine triphosphates (GTPs) are hydrolyzed on EF-Tu for every bound aa-tRNA. This was rationalized by the notion of an extended ternary complex, consisting of two EF-Tu.GTPs bound to a single aa-tRNA. In this work, we combine fast kinetics with RNase A protection experiments to measure the stoichiometry between EF-Tu.GTP and aa-tRNA at 37 degrees C, where the binding is weak. We find a 2:1 stoichiometry between EF-Tu.GTP and aa-tRNA at 37 degrees C, but at 0 degree C, under otherwise similar conditions, the stoichiometry of the complex is close to 1:1. These results suggest alternative pathways for aa-tRNA binding to ribosomes, since two GTPs are hydrolyzed per peptide bond at both temperatures. At 37 degrees C, aa-tRNA enters the A-site in a pentameric complex with two EF-Tu's on which two GTPs are hydrolyzed in synchrony, when cognate codon-anticodon contact is established. This pentameric model also explains how two GTPs can be hydrolyzed on EF-Tu, without rejection of 50% of the cognate aa-tRNAs in proofreading. At 0 degree C, in contrast, two ordinary ternary complexes may form a pentameric complex on, rather than off, the ribosome. When the two EF-Tu bound GTPs are hydrolyzed, one aa-tRNA enters the A-site, and the other dissociates to the free state.

Escherichia coli↗

PubMed1995

Two GTPs are consumed on EF-Tu per peptide bond in poly(Phe) synthesis, in spite of switching stoichiometry of the EF-Tu.aminoacyl-tRNA complex with temperature.

Recent observations indicate that the stoichiometry for the complex between EF-Tu.GTP and aminoacyl-tRNA (aa-tRNA) changes with temperature. At 37 degrees C two EF-Tu.GTPs bind one aa-tRNA in an extended ternary complex, but at 0 degrees C the complex has 1:1 stoichiometry. However, the present experiments show that there are two GTPs hydrolyzed on EF-Tu per peptide bond in poly(Phe) synthesis at 37 degrees C as well as at 0 degrees C. This indicates two different pathways for the enzymatic binding of aa-tRNA to the A-site on the ribosome.

Escherichia coli↗

PubMed1994

Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.

Binding Sites↗

PubMed1994

Mutations in 23 S ribosomal RNA perturb transfer RNA selection and can lead to streptomycin dependence.

Escherichia coli ribosomes with a G to C transversion at position 2661 in 23 S ribosomal RNA are more accurate in tRNA selection than wild-type ribosomes. This enhanced accuracy is due to improved initial selection of ternary complexes rather than proofreading of aminoacyl tRNAs. The 2661C mutation reduces the binding rate of cognate ternary complexes to the A-site. This binding rate deficiency becomes dramatic when ribosomes also harbour an S12 mutation with a streptomycin-resistant, hyperaccurate phenotype. In this case, severe loss of kinetic efficiency in EF-Tu function leads to cell death. Streptomycin restores viability by increasing the association rate of ternary complex to these doubly altered ribosomes. The binding rate of EF-G to 2661C ribosomes is also reduced while the translocation rate is unaffected.

Escherichia coli↗

PubMed1994

In vitro measurement of translation accuracy of ribosomes isolated from streptomycin-resistant mutant of Streptomyces granaticolor.

Accuracy of activity of ribosome isolated from UV-light-induced streptomycin-resistant R-21 mutant of Streptomyces granaticolor was measured in an E. coli-derived system translating poly(U) with a high rate and accuracy. Ribosomes from the R-21 mutant strain were shown to be resistant to streptomycin and about two-fold more accurate than those from the wild type. The mutant strain was found to be resistant to 1000 mg/L streptomycin (Stm) during vegetative growth while it sporulated on agar plates containing only up to 200 mg/L of Stm. The growth rate of the R-21 mutant in complex liquid medium was indistinguishable from that of the wild-type strain.

Drug Resistance, Microbial↗

PubMed1994

Two GTPs are hydrolysed on two molecules of EF-Tu for each elongation cycle during code translation.

A new experimental design has been used to determine the number of GTPs hydrolysed per peptide bond in EF-Tu function in a poly(U)-translation system. We find that two GTPs are consumed for every amino acid incorporated into the nascent poly(Phe)-chains, in accordance with previous findings with other techniques. These results necessitate a revision of current views concerning E coli translation; also new schemes for ribosome function are discussed.

Buffers↗

PubMed1992

Kinetic properties of Escherichia coli ribosomes with altered forms of S12.

E. coli ribosomes with alterations in S12 leading to streptomycin resistance (SmR), dependence (SmD) and pseudodependence (SmP) were studied with the quench-flow technique. Kinetic changes at the various steps of the elongation cycle were identified. The rate of hydrolysis of GTP in the ternary complex in the ribosomal A-site is decreased drastically in SmD and moderately in SmP in relation to wild-type ribosomes. Addition of streptomycin restores much of the wild-type behaviour. The SmD, SmP and SmR ribosomes have an enhanced GTP-hydrolysis idling reaction on EF-Tu, which is correlated with how aggressive proofreaders these ribosomes are in steady-state assays. We use our in vitro findings to discuss the in vivo physiology of these mutants as well as mechanistic features of E. coli translation.

Escherichia coli↗

PubMed1991

ms2i6A deficiency enhances proofreading in translation.

The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine. Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro. Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged. Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP. ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type. ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).

Codon↗

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↗