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Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account., (Copyright © 2014. Published by Elsevier Ltd.)

We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s(-1) with an apparent pKa value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others., (Copyright © 2015. Published by Elsevier Ltd.)

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K(d)versus 9.1-13.9 μM K(d) at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA(fMet) demonstrating that IF2 binds to initiator tRNA with submicromolar K(d) and that affinity is altered by the G nucleotides only slightly. This--in conjunction with earlier reports on IF2 interactions with fMet-tRNA(fMet) in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA(fMet) binding and GTP was suggested to strongly promote fMet-tRNA(fMet) binding--sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA(fMet) selection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a DeltaS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA(fMet), and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function., (Copyright 2010 Elsevier Ltd. All rights reserved.)

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 degrees C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 degrees C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (-868+/-25 and -577+/-23 cal mol(-1) K(-1), respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the gamma-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5'-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-A (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S x EF-G x guanosine 5'-[(betagamma)-imido]triphosphate x RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5'-[(betagamma)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.

In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the stop codons with different affinities and trigger the hydrolysis of the ester bond that links the polypeptide with the P-site tRNA. Cryo-electron microscopy (cryo-EM) results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center. These results are similar to those obtained for RF2, but there is an important difference in how the factors bind to protein L11, which forms part of the GTPase-associated center of the large ribosomal subunit. The difference in the binding position, C-terminal domain for RF2 versus N-terminal domain for RF1, explains a body of L11 mutation studies that revealed differential effects on the activity of the two factors. Very recent data obtained with small-angle X-ray scattering now reveal that the solution structure of RF1 is open, as here seen on the ribosome by cryo-EM, and not closed, as seen in the crystal.

During mRNA translation, synonymous codons for one amino acid are often read by different isoaccepting tRNAs. The theory of selective tRNA charging predicts greatly varying percentages of aminoacylation among isoacceptors in cells starved for their common amino acid. It also predicts major changes in tRNA charging patterns upon concentration changes of single isoacceptors, which suggests a novel type of translational control of gene expression. We therefore tested the theory by measuring with Northern blots the charging of Leu-tRNAs in Escherichia coli under Leu limitation in response to over expression of tRNA(GAG)(Leu). As predicted, the charged level of tRNA(GAG)(Leu) increased and the charged levels of four other Leu isoacceptors decreased or remained unchanged, but the charged level of tRNA(UAG)(Leu) increased unexpectedly. To remove this apparent inconsistency between theory and experiment we postulated a previously unknown common codon for tRNA(GAG)(Leu) and tRNA(UAG)(Leu). Subsequently, we demonstrated that the tRNA(GAG)(Leu) codon CUU is, in fact, read also by tRNA(UAG)(Leu), due to a uridine-5-oxyacetic acid modification.

A ribosome stalled on a truncated mRNA in the eubacterial cell can be rescued by tmRNA via a process called trans-translation. We demonstrate here that release of truncated mRNAs from stalled ribosomes accelerates significantly already after trans-peptidation following tmRNA binding to the ribosome. However, rapid release of truncated mRNA requires EF-G-dependent translocation of peptidyl-tmRNA from the A to the P site of the ribosome. We show also that the rate of mRNA release before and after peptidyl-tmRNA translocation correlates well with the rate of dissociation of deacylated tRNA, indicating that mRNA is retained on the ribosome mainly through codon:anticodon interaction with tRNA. The rate of mRNA release is reduced for mRNAs with strong Shine-Dalgarno (SD)-like sequences in the vicinity of the truncation site as well as for mRNAs with long 3' extensions downstream from the P-site codon. The reduced rate of release in the former case was due to a persisting SD-anti SD interaction between mRNA and the ribosome.

Understanding and controlling the interactions between foreign materials and cytoplasmic proteins is key for the design of intracellular probes, and for uncovering mechanisms of micro and nanoparticle toxicity. Here we examine these interactions by characterizing protein adsorption from cell extracts to a range of micron and sub-micron particles, and by measuring the Brownian motions of particles in live cells and reconstituted networks as an in situ measure of association. Testing SiO2, TiO2 and polystyrene particles with varying surface carboxylation, together with protein and polyethylene glycol surface coatings, we find that cellular associations and protein binding both strongly depend on particle surface chemistry. Cytoskeletal proteins, most notably actin and intermediate filament family members, are among the proteins most concentrated on the surfaces of all particles tested. The nanoscale movements of microinjected particles that primarily bind vimentin intermediate filaments are larger than particles that can also bind actin. This difference disappears when the same particles are endocytosed, suggesting that endocytic membranes mask particle surfaces. We discovered one brand of carboxylated SiO2 particles that is remarkably resistant to protein binding in extracts. By coupling the actin binding molecule phalloidin to these particles, we converted their surface from non-binding to actin-binding. We illustrate the efficacy of the conversion in reconstituted actin gels.

The notion that the ribosome is dynamic has been supported by various biochemical techniques, as well as by differences observed in high-resolution structures of ribosomal complexes frozen in various functional states. Yet, the mechanisms and extent of rRNA dynamics are still largely unknown. We have used a novel, fast chemical-modification technique to provide time-resolved details of 16 S rRNA structural changes that occur as bridges are formed between the ribosomal subunits as they associate. Association of different 16 S rRNA regions was found to be a sequential, multi-step process involving conformational rearrangements within the 30 S subunit. Our results suggest that key regions of 16 S rRNA, necessary for decoding and tRNA A-site binding, are structurally altered in a time-dependent manner by association with the 50 S ribosomal subunits.

tmRNA targets ribosomes, stalled either on truncated mRNAs or on mRNAs with slowly read sense or stop codons, tags the newly synthesized peptide chains for degradation and allows for their release by a class-1 release factor. We have studied in vitro how the rate of trans-transfer of a peptide from the P-site tRNA to tmRNA and the efficiency by which tmRNA competes with peptide release factors depend on the length of the mRNA downstream from the P-site. We show that the rate and efficiency of tmRNA action decrease rapidly with increasing down stream length and approach zero when it exceeds 15 bases. We demonstrate that tmRNA action is strongly stimulated by RelE cleavage of mRNA in the A site. We conclude that tmRNA action in vivo must always be preceded by mRNA truncation, and suggest that cleavage of ribosome bound mRNAs is a common element in different bacterial stress responses.

The macrolide-lincosamide-streptogramin B class (MLS) of antibiotics contains structurally different but functionally similar drugs, that all bind to the 50S ribosomal subunit. It has been suggested that these compounds block the path by which nascent peptides exit the ribosome. We have studied the mechanisms of action of four macrolides (erythromycin, josamycin, spiramycin and telithromycin), one lincosamide (clindamycin) and one streptogramin B (pristinamycin IA). All these MLS drugs cause dissociation of peptidyl-tRNA from the ribosome. Josamycin, spiramycin and clindamycin, that extend to the peptidyl transferase center, cause dissociation of peptidyl-tRNAs containing two, three or four amino acid residues. Erythromycin, which does not reach the peptidyl transferase center, induces dissociation of peptidyl-tRNAs containing six, seven or eight amino acid residues. Pristinamycin IA causes dissociation of peptidyl-tRNAs with six amino acid residues and telithromycin allows polymerisation of nine or ten amino acid residues before peptidyl-tRNA dissociates. Our data, in combination with previous structural information, suggest a common mode of action for all MLS antibiotics, which is modulated by the space available between the peptidyl transferase center and the drug.

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., (Copyright 2001 Academic Press.)

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., (Copyright 2000 Academic Press.)

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains., (Copyright 1999 Academic Press.)

Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis., (Copyright 1999 Academic Press.)

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay., (Copyright 1999 Academic Press.)

An in vitro assay in which terminating Escherichia coli ribosomes with different stop signals in the A-site compete for a limited amount of a release factor (RF1 or RF2) has been used to estimate the relative termination efficiencies at stop codons with different adjacent downstream nucleotides. The assay allows direct measurements of relative kcat/Km parameters for the productive association of release factors to ribosomes. The kcat/Km parameter is larger for UAA(U) than for UAA(C) programmed ribosomes and the difference in kcat/Km is much larger for RF2 (about 80%) than for RF1 (about 30%). These differences in the kcat/Km parameter are not affected by the addition of release factor RF3. The only discernible effect of RF3 is a considerable acceleration of RF1/2 recycling.The estimated kcat/Km parameters correlate well with the affinities of release factors for ribosomes programmed with different stop signals. These affinities were estimated from the extent of inhibition of ribosomal recycling by high concentrations of release factors in the absence of release factor RF3. The affinity for RF2 depends on the immediate downstream context of the stop codon in the translated mRNA and is about three times higher for UAA(U) than for UAA(C). The corresponding difference in affinities for RF1 is twofold. For all stop signals studied, the estimated affinity of RF2 for terminating ribosomes is much lower than that of RF1. It is also striking that the affinity of ribosomes for a chromosomally expressed RF2 is at least three times higher than for RF2 isolated from an overproducing E. coli strain., (Copyright 1998 Academic Press)

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed., (Copyright 1998 Academic Press)

A model of ColE1 copy number control has been developed where molecular details of replication are connected both to segregational stability and metabolic burden. Efficient replication control reduces copy number variation and increases segregational stability for a given average copy number. Copy number variation is predicted to depend on the type of inhibition mechanism as well as RNA I and RNA II turnover rate constants. It is shown that when both RNA I and RNA II transcription frequencies and the rate constant for degradation of free RNA I are very large, a hyperbolic inhibition mechanism must compensate with a 1.4 times greater average copy number to obtain the same segregational stability as an exponential inhibition mechanism. How sensitively the replication frequency responds to changes in RNA I concentration depends on the type of inhibition mechanism and the number of attempts to form an RNA II replication primer per plasmid and cell cycle. If RNA I is too stable, it will not follow changes in plasmid concentration closely, and when the transcription frequency for RNA I is only slightly higher than for RNA II, RNA I concentration becomes randomized. In both these cases, the proportionality between the single cell RNA I and plasmid concentrations is lost and this impairs copy number control. Thresholds in the rate for degradation of free RNA I as well as in RNA I and RNA II transcription frequencies have been computed, where an increase in these rate constants has a negligible effect on segregational stability but a corresponding decrease leads to segregational disaster. This indicates that there exists a well defined optimal set of rate constants where the regulation system works well without excessive metabolic load. A number of new experiments are suggested to address features of particular importance for the evolution of ColE1 copy number control.

The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on the concentrations of release factor RF1 and the ribosome recycling factor (RRF) has been studied in vitro. High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduction depended on stop codon context. The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF concentration. This indicates that RF1 and RRF have mutually exclusive and perhaps overlapping binding sites on the ribosome. Addition of release factor RF3 to the translation system abolishes the inhibitory effect of high RF1 concentration and increases the overall rate of ribosome recycling. These data can be explained by a three-step model for termination where the first step is RF1-promoted hydrolysis of peptidyl-tRNA. The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3. The third step, catalysed by RRF and elongation factor G, leads to mobility of the ribosome on mRNA allowing it to enter a further round of translation. In the absence of RF3, RF1 can re-associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, preventing RRF from entering the ribosomal A-site and thereby inhibiting ribosomal recycling. The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vitro experiments., (Copyright 1997 Academic Press Limited.)

Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (kcat/KM) for both EF-Tu and EF-G binding. In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are significantly increased by the A to U transversion. The U1067 mutation impairs EF-Tu function more than EF-G function. The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu.GDP release from the mutated ribosomes. There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition. With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu., (Copyright 1997 Academic Press Limited.)

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.

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.

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.

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.

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).

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.

To study the risk of developing rubeosis iridis following vitrectomy, the use of preoperative iris fluorescein angiography was prospectively investigated on patients with diabetes mellitus, without any clinically detectable iris or angle neovascularization. With increasing severity of fluorescein leakage, the risk of iris neovascularization with or without glaucoma, six months after vitreous surgery, is significantly increased. It was also determined whether postoperative steroid therapy (periocular triamcinolone acetonide and topical prednisolone phosphate) reduces the risk of developing iris neovascularization after vitrectomy. Controlled and treated patient eyes did not differ significantly in the degree of rubeosis. However, neovascular glaucoma statistically occurred more frequently in the nonsteroid group.