Biomedical subjects

C G Kurland

Publications and source records attributed to C G Kurland.

At least 19 recordsLinked to original sources

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↗

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↗

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↗

PubMed1990

Growth rate dependence of global amino acid composition.

The global amino acid composition of bacteria growing in different media has been studied. The data reveal significant changes in the amino acid composition in the growth rate range between 0.5 and 2.1 doublings per hour at 37 degrees C. The changes are consistent with a progressive simplification of the protein population and mRNA pools as the growth rates increase.

Amino Acids↗

PubMed1990

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

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

Escherichia coli↗

PubMed1990

Growth rate dependence of transfer RNA abundance in Escherichia coli.

We have tested the predictions of a model that accounts for the codon preferences of bacteria in terms of a growth maximization strategy. According to this model the tRNA species cognate to minor and major codons should be regulated differently under different growth conditions: the isoacceptors cognate to major codons should increase at fast growth rates while those cognate to minor codons should decrease at fast growth rates. We have used a quantitative Northern blotting technique to measure the abundance of the methionine and the leucine isoacceptor families over growth rates ranging from 0.5 to 2.1 doublings per hour. Five tRNA species that are cognate to major codons (tRNA(eMet), tRNA(1fMet), tRNA(2fMet), tRNA(1Leu) and tRNA(3Leu) increase both as a relative fraction of total tRNA and in absolute concentration with increasing growth rates. Three tRNA species that are cognate to minor codons (tRNA(2Leu), tRNA(4Leu) and tRNA(5Leu) decrease as a relative fraction of total RNA and in absolute concentration with increasing growth rates. These data suggest that the abundances of groups of tRNA species are regulated in different ways, and that they are not regulated simply according to isoacceptor specificity. In particular, the data support the growth optimization model for codon bias.

Base Sequence↗

PubMed1990

Ribosomal RNA and protein mutants resistant to spectinomycin.

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

Escherichia coli↗