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Biomedical subjects

M Ehrenberg

Publications and source records attributed to M Ehrenberg.

At least 19 recordsLinked to original sources
PubMed2006

Rate, accuracy and cost of ribosomes in bacterial cells.

Recent biochemical data on the rate of peptidyl-transfer and missense error levels associated with the E. coli ribosome in conjunction with direct measurements of diffusion constants for proteins in the E. coli cell have been used to discuss protein synthesis in the living E. coli cell in the perspective of a previously developed maximal fitness theory. With these improved experimental parameters, i.e. kcat approximately 50 s(-1) for protein elongation and kcat/KM approximately 4 microM(-1) s(-1) for cognate ternary complex binding to the ribosomal A site, theory predicts the experimentally observed variations in protein elongation rate, ribosome and ternary complex concentrations with varying quality of the growth medium. The theoretically predicted average missense error level is close the error levels estimated in vitro for special isoacceptor combinations, i.e. error levels about 1 per million. The future prospect of extensive integration of biochemistry, cell physiology and population genetics is discussed in the light of the maximal fitness theory and other, similar, theoretical approaches.

Bacteria↗
PubMed2004

Spontaneous separation of bi-stable biochemical systems into spatial domains of opposite phases.

Bi-stable chemical systems are the basic building blocks for intracellular memory and cell fate decision circuits. These circuits are built from molecules, which are present at low copy numbers and are slowly diffusing in complex intracellular geometries. The stochastic reaction-diffusion kinetics of a double-negative feedback system and a MAPK phosphorylation-dephosphorylation system is analysed with Monte-Carlo simulations of the reaction-diffusion master equation. The results show the geometry of intracellular reaction compartments to be important both for the duration and the locality of biochemical memory. Rules for when the systems lose global hysteresis by spontaneous separation into spatial domains in opposite phases are formulated in terms of geometrical constraints, diffusion rates and attractor escape times. The analysis is facilitated by a new efficient algorithm for exact sampling of the Markov process corresponding to the reaction-diffusion master equation.

Adaptation, Physiological↗
PubMed2003

Free RNA polymerase and modeling global transcription in Escherichia coli.

Growth rate-dependent changes in the cytoplasmic concentration of free functional RNA polymerase, [R(f)], affect the activity of all bacterial genes. Since [R(f)] is not accessible to direct experimental quantitation, it can only be found indirectly from an evaluation of promoter activity data. Here, a theory has been derived to calculate [R(f)] from the concentrations of total RNA polymerase and promoters in a model system with known Michaelis-Menten constants for the polymerase-promoter interactions. The theory takes transcript lengths and elongation rates into account and predicts how [R(f)] changes with varying gene dosages. From experimental data on total concentrations of RNA polymerase and kinetic properties of different classes of promoters, the theory was developed into a mathematical model that reproduces the global transcriptional control in Escherichia coli growing at different rates. The model allows an estimation of the concentrations of free and DNA-bound RNA polymerase, as well as the partitioning of RNA polymerase into mRNA and stable RNA synthesizing fractions. According to this model, [R(f)] is about 0.4 and 1.2 microM at growth rates corresponding to 1.0 and 2.5 doublings/h, respectively. The model accurately reflects a number of further experimental observations and suggests that the free RNA polymerase concentration increases with increasing growth rate.

DNA, Bacterial↗
PubMed2002

Kinetic properties of rrn promoters in Escherichia coli.

How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter-lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [R(f)], and the ratio of the Michaelis-Menten parameters, V(max)/K(m) (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD(460)) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD(460) and corresponds to an intracellular concentration of about 50 microM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.

DNA, Ribosomal↗
PubMed2001

Comparison of repressor and transcriptional attenuator systems for control of amino acid biosynthetic operons.

In bacteria, expression from amino acid biosynthetic operons is transcriptionally controlled by two main mechanisms with principally different modes of action. When the supply of an amino acid is in excess over demand, its concentration will be high and when the supply is deficient the amino acid concentration will be low. In repressor control, such concentration variations in amino acid pools are used to regulate expression from the corresponding amino acid synthetic operon; a high concentration activates and a low concentration inactivates repressor binding to the operator site on DNA so that initiation of transcription is down or up-regulated, respectively. Excess or deficient supply of an amino acid also speeds or slows, respectively, the rate by which the ribosome translates mRNA base triplets encoding this amino acid. In attenuation of transcription, it is the rate by which the ribosome translates such "own" codons in the leader of an amino acid biosynthetic operon that decides whether the RNA polymerase will continue into the operon, or whether transcription will be aborted (attenuated). If the ribosome rate is fast (excess synthesis of amino acid), transcription will be terminated and if the rate is slow (deficient amino acid supply) transcription will continue and produce more messenger RNAs. Repressor and attenuation control systems have been modelled mathematically so that their behaviour in living cells can be predicted and their system properties compared. It is found that both types of control systems are unexpectedly sensitive when they operate in the cytoplasm of bacteria. In the repressor case, this is because amino acid concentrations are hypersensitive to imbalances between supply and demand. In the attenuation case, the reason is that the rate by which ribosomes translate own codons is hypersensitive to the rate by which the controlled amino acid is synthesised. Both repressor and attenuation mechanisms attain close to Boolean properties in vivo: gene expression is either fully on or fully off except in a small interval around the point where supply and demand of an amino acid are perfectly balanced.Our results suggest that repressors have significantly better intracellular performance than attenuator mechanisms. The reason for this is that repressor, but not attenuator, mechanisms can regulate expression from biosynthetic operons also when transfer RNAs are fully charged with amino acids so that the ribosomes work with maximal speed.

Acylation↗
PubMed2001

A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3.

The mechanism by which peptide release factor RF3 recycles RF1 and RF2 has been clarified and incorporated in a complete scheme for translation termination. Free RF3 is in vivo stably bound to GDP, and ribosomes in complex with RF1 or RF2 act as guanine nucleotide exchange factors (GEF). Hydrolysis of peptidyl-tRNA by RF1 or RF2 allows GTP binding to RF3 on the ribosome. This induces an RF3 conformation with high affinity for ribosomes and leads to rapid dissociation of RF1 or RF2. Dissociation of RF3 from the ribosome requires GTP hydrolysis. Our data suggest that RF3 and its eukaryotic counterpart, eRF3, have mechanistic principles in common.

Guanine Nucleotide Exchange Factors↗
PubMed2001

The mRNA cap structure stimulates rate of poly(A) removal and amplifies processivity of degradation.

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive, and cap-interacting 3' exonuclease. We have studied how the m7G(5')ppp(5')G cap structure affects the activity of PARN. It is shown that the cap has four distinct effects: (i) It stimulates the rate of deadenylation if provided in cis; (ii) it inhibits deadenylation if provided at high concentration in trans; (iii) it stimulates deadenylation if provided at low concentration in trans; and (iv) it increases the processivity of PARN when provided in cis. It is shown that the catalytic and cap binding sites on PARN are separate. The important roles of the 7-methyl group and the inverted guanosine residue of the cap are demonstrated. An active deadenylation complex, consisting of the poly(A)-tailed RNA substrate and PARN, has been identified. Complex formation does not require a cap structure on the RNA substrate. The multiple effects of cap are all accounted for by a simple, kinetic model that takes the processivity of PARN into account.

Chromatography, Thin Layer↗
PubMed2000

A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation.

A post-translational modification affecting the translation termination rate was identified in the universally conserved GGQ sequence of release factor 2 (RF2) from Escherichia coli, which is thought to mimic the CCA end of the tRNA molecule. It was shown by mass spectrometry and Edman degradation that glutamine in position 252 is N:(5)-methylated. Overexpression of RF2 yields protein lacking the methylation. RF2 from E.coli K12 is unique in having Thr246 near the GGQ motif, where all other sequenced bacterial class 1 RFs have alanine or serine. Sequencing the prfB gene from E.coli B and MRE600 strains showed that residue 246 is coded as alanine, in contrast to K12 RF2. Thr246 decreases RF2-dependent termination efficiency compared with Ala246, especially for short peptidyl-tRNAs. Methylation of Gln252 increases the termination efficiency of RF2, irrespective of the identity of the amino acid in position 246. We propose that the previously observed lethal effect of overproducing E.coli K12 RF2 arises through accumulating the defects due to lack of Gln252 methylation and Thr246 in place of alanine.

Alanine↗
PubMed2000

Stochastic focusing: fluctuation-enhanced sensitivity of intracellular regulation.

Many regulatory molecules are present in low copy numbers per cell so that significant random fluctuations emerge spontaneously. Because cell viability depends on precise regulation of key events, such signal noise has been thought to impose a threat that cells must carefully eliminate. However, the precision of control is also greatly affected by the regulatory mechanisms' capacity for sensitivity amplification. Here we show that even if signal noise reduces the capacity for sensitivity amplification of threshold mechanisms, the effect on realistic regulatory kinetics can be the opposite: stochastic focusing (SF). SF particularly exploits tails of probability distributions and can be formulated as conventional multistep sensitivity amplification where signal noise provides the degrees of freedom. When signal fluctuations are sufficiently rapid, effects of time correlations in signal-dependent rates are negligible and SF works just like conventional sensitivity amplification. This means that, quite counterintuitively, signal noise can reduce the uncertainty in regulated processes. SF is exemplified by standard hyperbolic inhibition, and all probability distributions for signal noise are first derived from underlying chemical master equations. The negative binomial is suggested as a paradigmatic distribution for intracellular kinetics, applicable to stochastic gene expression as well as simple systems with Michaelis-Menten degradation or positive feedback. SF resembles stochastic resonance in that noise facilitates signal detection in nonlinear systems, but stochastic resonance is related to how noise in threshold systems allows for detection of subthreshold signals and SF describes how fluctuations can make a gradual response mechanism work more like a threshold mechanism.

Animals↗
PubMed2000

Random signal fluctuations can reduce random fluctuations in regulated components of chemical regulatory networks.

Many intracellular components are present in low copy numbers per cell and subject to feedback control. We use chemical master equations to analyze a negative feedback system where species X and S regulate each other's synthesis with standard intracellular kinetics. For a given number of X-molecules, S-variation can be significant. We show that this signal noise does not necessarily increase X-variation as previously thought but, surprisingly, can be necessary to reduce it below a Poissonian limit. The principle resembles Stochastic Resonance in that signal noise improves signal detection.

DNA Replication↗
PubMed2000

Origins of minigene-dependent growth inhibition in bacterial cells.

The expression of very short open reading frames in Escherichia coli can lead to the inhibition of translation and an arrest in cell growth. Inhibition occurs because peptidyl-tRNA hydrolase fails to recycle sufficiently rapidly peptidyl-tRNA released from ribosomes at the stop signal in competition with normal termination, causing starvation for essential species of tRNA. Previous studies have shown that the last sense codon, the strength of the Shine-Dalgarno sequence and the nature and context of the stop codon affect the toxicity associated with mini-gene expression. Here, several important parameters are studied as a function of the length of the mini-gene coding sequence. The rate of peptidyl-tRNA drop-off catalysed by translation factors decreases dramatically for peptides longer than a hexamer. The probability that ribosomes recycle without dissociation of the mini-gene mRNA varies strongly with the length of the coding sequence. The peptidyl-tRNA hydrolase rap mutant, unlike the wild-type enzyme, is highly sensitive to the length and sequence of the peptide. Together, these parameters explain the length dependence of mini-gene toxicity.

Bacterial Proteins↗
PubMed2000

Molecular clocks reduce plasmid loss rates: the R1 case.

Plasmids control their replication so that the replication frequency per plasmid copy responds to the number of plasmid copies per cell. High sensitivity amplification in replication response to copy number deviations generally reduces variation in copy numbers between different single cells, thereby reducing the plasmid loss rate in a cell population. However, experiments show that plasmid R1 has a gradual, insensitive replication control predicting considerable copy number variation between single cells. The critical step in R1 copy number control is regulation of synthesis of a rate-limiting cis-acting replication protein, RepA. De novo synthesis of a large number of RepA molecules is required for replication, suggesting that copy number control is exercised at multiple steps. In this theoretical kinetic study we analyse R1 multistep copy number control and show that it results in the insensitive replication response found experimentally but that it at the same time effectively prohibits the existence of only one plasmid copy in a dividing cell. In combination with the partition system of R1, this can lead to very high segregational stability. The R1 control mechanism is compared to the different multistep copy number control of plasmid ColE1 that is based on conventional sensitivity amplification. This implies that while copy number control for ColE1 efficiently corrects for fluctuations that have already occurred, R1 copy number control prevents their emergence in cells that by chance start their cycle with only one plasmid copy. We also discuss how regular, clock-like, behaviour of single plasmid copies becomes hidden in experiments probing collective properties of a population of plasmid copies because the individual copies are out of phase. The model is formulated using master equations, taking a stochastic approach to regulation, but the mathematical formalism is kept to a minimum and the model is simplified to its bare essence. This simplicity makes it possible to extend the analysis to other replicons with similar design principles.

Bacteriocin Plasmids↗
PubMed2000

The accuracy of codon recognition by polypeptide release factors.

The precision with which individual termination codons in mRNA are recognized by protein release factors (RFs) has been measured and compared with the decoding of sense codons by tRNA. An Escherichia coli system for protein synthesis in vitro with purified components was used to study the accuracy of termination by RF1 and RF2 in the presence or absence of RF3. The efficiency of factor-dependent termination at all sense codons differing from any of the three stop codons by a single mutation was measured and compared with the efficiency of termination at the three stop codons. RF1 and RF2 discriminate against sense codons related to stop codons by between 3 and more than 6 orders of magnitude. This high level of accuracy is obtained without energy-driven error correction (proofreading), in contrast to codon-dependent aminoacyl-tRNA recognition by ribosomes. Two codons, UAU and UGG, stand out as hotspots for RF-dependent premature termination.

Codon, Terminator↗
PubMed2000

Fluctuations and quality of control in biological cells: zero-order ultrasensitivity reinvestigated.

Living cells differ from most other chemical systems in that they involve regulation pathways that depend very nonlinearly on chemical species that are present in low copy numbers per cell. This leads to a variety of intracellular kinetic phenomena that elude macroscopic modeling, which implicitly assumes that cells are infinitely large and fluctuations negligible. It is of particular importance to assess how fluctuations affect regulation in cases where precision and reliability are required. Here, taking finite cell size and stochastic aspects into account, we reinvestigate theoretically the mechanism of zero-order ultrasensitivity for covalent modification of target enzymes ( Proc. Natl. Acad. Sci. USA. 78:6840-6844). Macroscopically, this mechanism can produce a very sharp transition in target concentrations for very small changes in the activity of the converter enzymes. This study shows that the transition is much more gradual in a finite cell or a population of finite cells. It also demonstrates that the switch is exactly analogous to a thermodynamic phase transition and that ultrasensitivity is inevitably coupled to random ultravariation. As a consequence, the average response in a large population of cells will often be much more gradual than predicted from macroscopic descriptions.

Cell Physiological Phenomena↗
PubMed2000

Fluctuations in repressor control: thermodynamic constraints on stochastic focusing.

The influence of fluctuations in molecule numbers on genetic control circuits has received considerable attention. The consensus has been that such fluctuations will make regulation less precise. In contrast, it has more recently been shown that signal fluctuations can sharpen the response in a regulated process by the principle of stochastic focusing (SF) (, Proc. Natl. Acad. Sci. USA. 97:7148-7153). In many cases, the larger the fluctuations are, the sharper is the response. Here we investigate how fluctuations in repressor or corepressor numbers can improve the control of gene expression. Because SF is found to be constrained by detailed balance, this requires that the control loops contain driven processes out of equilibrium. Some simple and realistic out-of-equilibrium steps that will break detailed balance and make room for SF in such systems are discussed. We conclude that when the active repressors are controlled by corepressor molecules that display large ("coherent") number fluctuations or when corepressors can be irreversibly removed directly from promoter-bound repressors, the response in gene activity can become significantly sharper than without intrinsic noise. A simple experimental design to establish the possibility of SF for repressor control is suggested.

DNA↗
PubMed2000

Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination.

Early studies provided evidence that peptide-chain release factors (RFs) bind to both ribosomal subunits and trigger translation termination. Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical evidence for the involvement of rRNA. Particularly absent is direct evidence for a role of a large subunit rRNA in RF binding. Previously we demonstrated in vitro that mutations in Escherichia coli rRNAs, known to cause nonsense codon readthrough in vivo, reduce the efficiency of RF2-driven catalysis of peptidyl-tRNA hydrolysis. This reduction was consistent with the idea that in vivo defective termination at the mutant ribosomes contributes to the readthrough. Nevertheless, other explanations were also possible, because still missing was essential biochemical evidence for that idea, namely, decrease in productive association of RFs with the mutant ribosomes. Here we present such evidence using a new realistic in vitro termination assay. This study directly supports in vivo involvement in termination of conserved rRNA regions that also participate in other translational events. Furthermore, this study provides the first strong evidence for involvement of large subunit rRNA in RF binding, indicating that the same rRNA region interacts with factors that determine both elongation and termination of translation.

Base Sequence↗