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C G Kurland

Publications and source records attributed to C G Kurland.

At least 19 recordsLinked to original sources
PubMed1996

Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates.

We have used two-dimensional polyacrylamide gel electrophoresis to fractionate tRNAs from Escherichia coli. A sufficiently high degree of resolution was obtained for 44 out of 46 tRNA species in E. coli to be resolved into individual electrophoretic components. These isolated components were identified by hybridization to tRNA-specific oligonucleotide probes. Systematic measurements of the abundance of each individual tRNA isoacceptor in E. coli, grown at rates varying from 0.4 to 2.5 doublings per hour, were made with the aid of this electrophoretic protocol. We find that there is a biased distribution of the tRNA abundance at all growth rates, and that this can be roughly correlated with the values of codon frequencies in the mRNA pools calculated for bacteria growing at different rates. The tRNA species cognate to abundant codons increase in concentration as the growth rate increases but not as dramatically as might be anticipated. The levels of most of the tRNA isoacceptors cognate to less abundant codons remain unchanged with increasing growth rates. The result of these changes in tRNA abundance is that the relative increase in the amounts of major tRNA species in the bacteria growing at the fastest growth rates is more modest than previous estimates from this laboratory suggested. Furthermore, a systematic error in previous estimates of ribosomal RNA content of the bacteria has been detected. This will account for the quantitative discrepancies between the previous and the present data for tRNA abundance.

Base Sequence↗
PubMed1995

Ribosome mutants with altered accuracy translate with reduced processivity.

We have determined whether or not there is a correlation between the accuracy of tRNA selection and the processivity of translation by mutant ribosomes. In our assay the estimation of processivity was made from a system in which transcription is dependent on T7 RNA polymerase in order to eliminate the influence of transcriptional polarity on our measurements. We found that the processivity of translation is reciprocally correlated to the degree with which mutant ribosomes restrict nonsense suppression events. This is observed when transcription is from both T7 RNA polymerase and Escherichia coli RNA polymerase. In other words, we have confirmed the expected correlation between increased accuracy of tRNA selection and decreased processivity of translation. No such monotonic correlation was obtained with ribosome ambiguity (ram) mutants, though all of these were less processive than wild-type ribosomes. In addition, streptomycin was found to simultaneously stimulate tRNA selection errors and processivity of translation for a mutant that is partially dependent on streptomycin. These data suggest that the growth rates of ribosome mutants are significantly decreased by the degree to which their processivity in translation is affected. Furthermore, we suggest that streptomycin dependence is a phenomenon reflecting the interplay of the restrictive mutation and antibiotic on the processivity of translation.

DNA-Directed RNA Polymerases↗
PubMed1995

Unusual organization of the rRNA genes in Rickettsia prowazekii.

We describe here the organization of the rRNA genes in Rickettsia prowazekii. In this organism, the 23S and the 5S rRNA genes are tightly linked to each other, whereas the 16S rRNA gene is separated from this cluster. The 23S-5S unit is preceded by the methionyl-tRNAfMet formyltransferase gene.

Acyltransferases↗
PubMed1995

Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction.

We attempted to test the idea that the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration. In order to change the overall codon composition of the messenger pool, we have expressed in E. coli lacZ with the aid of T7 RNA polymerase so that their respective gene products individually accounted for 30% of the total bacterial protein. Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that we studied. Instead, we find that there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity. After either of the test genes is maximally induced, there is a growing fraction of protein synthesis invested in beta-galactosidase or delta tufB that is matched by a comparable decrease of the fraction of normal protein synthesis. We have also observed enhanced accumulation of two heat shock proteins during overexpression. Finally, after several hours of overexpression of either test protein, the bacteria are no longer viable. These results are relevant to the practical problems of obtaining high expression levels for cloned proteins.

Bacterial Proteins↗
PubMed1994

The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli.

The number of molecules of release factor-1 (RF-1) and release factor-2 (RF-2) per Escherichia coli cell grown at various rates was determined using quantitative Western blotting of total solubilized cell protein. The number of RF-1 molecules per cell increased from 1200 to 4900, and of RF-2 from 5900 to 24,900 as growth rates increased from 0.3 to 2.4 doublings per hour. The cellular concentration of the release factors, and therefore efficient termination of protein synthesis is maintained by the increased expression of both RFs as growth rate increases. The expression of both release factors RF-1 and RF-2 is co-ordinated with that of the rest of the translational apparatus, although the increases are less for RF than that for the ribosomes under the same conditions. A significant proportion of the RF pool was found associated with the ribosome fraction. The percentage of ribosomes containing an RF molecule increased from 21 to 33% as the translational rate increased over the growth rate range. Since the cellular concentration of the release factors and their specific activity does not vary significantly with growth rate, this can not provide for an increase in the rate at any of the steps of termination. The postulated strong stop signals, UAAU and UAAG, in genes that are highly expressed at fast growth rates, may result in an increase in the termination rate as a consequence of increased efficiency of decoding by RFs.

Cell Fractionation↗
PubMed1993

Growth-rate-dependent accumulation of twelve tRNA species in Escherichia coli.

We have previously shown that in Escherichia coli the accumulation of five leucine and three methionine tRNA species is regulated so that those tRNA species that translate major codons increase while those that translate minor codons decrease as the growth rate increases. Here, we have analyzed the growth-rate-dependence of another 12 tRNA species. We find that the level of three tRNA species cognate to the major glycine, proline and arginine codons, respectively, increase with increasing growth rates. Conversely, four tRNAs that are cognate to minor codons within the same amino acid families decrease with increasing growth rates. In addition, the glutamyl as well as the phenylalanyl isoacceptor species are accumulated in proportion to the content of these two amino acids in the proteins produced at different growth rates. In summary, the patterns of the growth-rate-dependence for the accumulation of these 17 tRNA species support the interpretation that the major codon preference is an arrangement to maximize the growth rates of bacteria in rich media by optimizing the kinetic efficiency of translation. In contrast, we find that three minor tRNA species cognate to two rare arginine codons and one minor glycine codon, respectively, increase with increasing growth rate. Such findings suggest that there are additional constraints on the accumulation of these tRNA species that may be distinct from those required to optimize the kinetic efficiency of translation.

Base Sequence↗
PubMed1993

Release factor-dependent false stops are infrequent in Escherichia coli.

We have estimated the frequency of release factor dependent events in which a sense codon is mistakenly translated as a stop codon. We refer to this event as a "false stop". In order to facilitate the measurement of false stop freqeuncies we have used a plasmid expression system to increase individually the cellular levels of release factor (RF) I a and of release factor (RF) 2. We were then able to measure the loss of translational processivity with the aid of a lacZ processivity assay at different concentrations of the release factors. We find that a 30- to 40-fold increase of the RF1 concentration reduces lacZ processivity from 0.6 to 0.3. Assuming that the processivity loss is due only to false stops and that the RF1 overproduction data can be extrapolated back linearly to the normal RF1 concentration in the cell, this corresponds to a false stop frequency close to 10(-5) per codon in the presence of normal amounts of RF1. Furthermore, a threefold increase of the RF2 concentrations had no measurable effect on the processivity of the lacZ gene. Our data suggest that false stops are relatively infrequent compared to the incidence of other translation errors.

Cloning, Molecular↗
PubMed1992

Thiolation of transfer RNA in Escherichia coli varies with growth rate.

We have used an affinity electrophoresis assay which when combined with Northern hybridization techniques permits us to estimate the degree of thiolation of individual tRNA species in Escherichia coli. We observe that the levels of 4-thio 2'(3')-uridine (4-thioU) in many but not all tRNAs varies dramatically at different bacterial growth rates: Five tRNAs are completely thiolated at all growth rates, while another eight tRNAs are incompletely thiolated and the fraction of the unthiolated form of these tRNA species increases as the growth rates increase. Transfer RNA(2Glu) contains 4-thioU as well as (methylamino)methyl-2-thio uridine (mnm(5)2-thioU). The level of mnm(5)2-thioU of tRNA(2Glu) is invariant with growth rate. Surprisingly, none of the thirteen tRNA species that we have studied is completely unmodified in all growth media. In particular, at the slowest growth rates every tRNA class that we have studied contains a form that has 4-thioU residues.

Base Sequence↗
PubMed1992

Evolution of mitochondrial genomes and the genetic code.

Mitochondrial genomes are clearly marked by a strong tendency towards reductive evolution. This tendency has been facilitated by the transfer of most of the essential genes for mitochondrial propogation and function to the nuclear genome. The most extreme examples of genomic simplification are seen in animal mitochondria, where there also are the greatest tendencies to codon reassignment. The reassignment of codons to amino acids different from those designated in the so called universal code is seen in part as an expression of the reduction of the number of genes used by these genomes to code for tRNA species. The driving force for the reductive evolution of mitochondrial genomes is identified with two population genetic effects which may also be operating on populations of parasites.

Animals↗
PubMed1992

Selection of laboratory wild-type phenotype from natural isolates of Escherichia coli in chemostats.

We have followed, in glucose-limited chemostats, the evolution of natural isolates of Escherichia coli possessing maximal growth rates of 0.48-1.43 doublings/h. Under these conditions a rapid-growth phenotype similar to that of standard laboratory wild-type strains was selected so that after 280 generations all of the cultures were characterized by bacteria with maximum growth rates close to 1.33 doublings/h. The growth yields of the natural isolates, on the other hand, were quite uniform and improved only slightly during the selection; it seems that the natural isolates are nearly maximally efficient at utilizing glucose. Some of the kinetic characteristics of ribosomes prepared from natural isolates vary markedly and in proportion to the growth rates of the original strains. After growth in glucose-limited chemostats, the ribosomes of all of the cultures become kinetically indistinguishable from those of laboratory wild-type bacteria. These observations confirm the interpretation that bacteria grown under normal laboratory conditions have been selected for maximum growth rates which demand maximum translation efficiency. In contrast, these characteristics do not seem to be strongly selected in the natural isolates.

Culture Media↗
PubMed1992

Translational accuracy and the fitness of bacteria.

There are two aspects of the relationship between translational accuracy and the fitness of bacteria that I hope have been clarified in this review. One is that the impact of translational errors on the fitness of bacteria depends very much on nutritional conditions. It would seem that bacterial populations have the capacity to respond to different growth opportunities by the selection of suitable variants. It is particularly surprising how few mutations seem to be required to transform a slowly growing natural isolate with inefficient as well as inaccurate ribosomes into a growth-optimized laboratory strain. It would not be suprising if the selection of the slow, natural isolate phenotype under starvation conditions is equally facile. Another aspect of the accuracy-fitness relationship worth emphasizing is the strong impact of processivity errors and the weak impact of missense errors on the structures of proteins as well as on the growth of cells. What has been learned about translation mechanisms up to now is really only a preliminary to what remains to be discovered about the movements of tRNA, mRNA, and ribosomal subunits that support the processivity of translation. It would be very useful to have more direct methods at hand with which to study these movements. Likewise, the availability of methods to measure processivity errors in natural isolates would help to round out our view of the variability of the ribosomal mechanisms in nature.

Bacteria↗
PubMed1991

Codon bias and gene expression.

The frequencies with which individual synonymous codons are used to code their cognate amino acids is quite variable from genome to genome and within genomes, from gene to gene. One particularly well documented codon bias is that associated with highly expressed genes in bacteria as well as in yeast; this is the so-called major codon bias. Here, it is suggested that the major codon bias is not an arrangement for regulating individual gene expression. Instead, the data suggest that this codon bias, which is correlated with a corresponding bias of tRNA abundance, is a global arrangement for optimizing the growth efficiency of cells. On the practical side, it is suggested that heterologous gene expression is not as sensitive to codon bias as previously thought, but that it is quite sensitive to other characteristics of the heterologous gene.

Codon↗
PubMed1991

Evidence for demand-regulation of ribosome accumulation in E coli.

We have determined the relative concentrations of ribosomes accumulated under different growth conditions for a number of translational mutants as well as for some natural isolates of Escherichia coli. The mutants are a tRNA modification mutant (miaA), a streptomycin resistant (SmR) and a streptomycin pseudodependent (SmP) mutant as well as two ribosome ambiguity (ram) mutants. The natural isolates used in this study are known to function with submaximal ribosome kinetics. The data show that for all the ribosome mutants the concentration of ribosomes relative to that in wild type bacteria increases when the growth rate decreases. A small increase is also seen in the natural isolates. In contrast, the miaA mutant shows no increase in ribosome concentration under the same slow growth conditions. The results suggest that bacteria with kinetically impaired ribosomes can to some extent increase the number of ribosomes accumulated under poor growth conditions in order to compensate for their slower function. We use this observation to explain in part how bacteria growing in natural environments can escape the strong selection for maximized growth rates and for optimized ribosomes that are characteristic of laboratory strains.

Culture Media↗
PubMed1991

An extreme codon preference strategy: codon reassignment.

We argue that in animal mitochondria codon reassignments, such as those for AGA and AGG from arginine to serine or of AUA from isoleucine to methionine, are the result of an interplay between biased mutational forces and selective ones. In particular, there is a marked tendency for animal mitochondria to have very small genomes and to minimize their investment in components required for gene expression. These tendencies are expressed as a reduction in the diversity of tRNA isoacceptor species. In our view, the pressure to simplify tRNA populations, together with mutational bias against certain codons, will account for the codon reassignments observed in animal mitochondria. A parallel to the major codon bias in microorganisms, which likewise tends to reduce the diversity of the tRNA isoacceptor populations under fast growth conditions, may be drawn. Therefore, we suggest that codon reassignments are usefully viewed as an extreme form of codon bias.

Base Composition↗
PubMed1990

Stoichiometry of elongation factor G function in translation.

A steady-state translation system has been used in vitro to measure the stoichiometry with which elongation factor G-GTP complexes are dissipated during polypeptide elongation. It has been possible to separate this dissipation from that associated with elongation factor Tu function. Our measurements for the wild-type as well as for two mutant variants indicate that there is one elongation factor G-GTP complex dissipated per peptide bond in the steady-state.

Carbon Radioisotopes↗
PubMed1990

Processivity errors of gene expression in Escherichia coli.

Not all ribosomes that initiate translation of an mRNA sequence will successfully complete it and produce a full-length protein product. By comparing the amounts of lacZ monomer and lacZ dimer protein expressed from a plasmid in a strictly controlled assay, we calculate a dimer to monomer ratio of 0.76. We interpret this to mean that ribosomes have a 76% chance of completing the synthesis of a beta-galactosidase polypeptide. The remaining 24% of the initiated chains end in processivity accidents. For the wild-type, premature RNA polymerase termination is found to account for roughly one-third of the processivity accidents. For the hyperaccurate SmP mutant, we observe a processivity of 0.28, but the presence of streptomycin improves this to 0.50. Thus, the hyperaccuracy with respect to missense substitutions for this mutant is accompanied by a reduced processivity. Addition of streptomycin increase the first error class and reduces the second one. This finding is relevant to the optimization of ribosome function and the growth performance of ribosome mutants.

Base Sequence↗
PubMed1990

Novel mutants of elongation factor G.

A novel mutant form of elongation factor G (EF-G) in Escherichia coli is described. This variant EF-G restricts reading frame errors by a factor of 2 to 3 in vivo at two different positions in a lacIZ fusion. In addition, a conventional fusidic acid resistant (fusR) mutant of EF-G was compared with the restrictive mutant. Both mutants were characterized in vitro in a steady-state poly(U) translating system. The data indicate that the restrictive EF-G variant has an altered interaction with the ribosome both in vivo and in vitro. In contrast, the conventional fusR variant is altered in its interaction with GTP, which is evident in vitro.

Drug Resistance, Microbial↗