Jonathan Gallant

Mistranslation in E. coli

Flagellin, the protomeric subunit of bacterial flagella, contains no cysteine. We have detected the incorporation of trace quantities of 35S-cysteine into flagellin, highly purified and then resolved by SDS polyacrylamide gel electrophoresis, to measure mistranslation in vivo. Under normal conditions, this value is about 6 X 10(-4) pmoles cysteine per pmole flagellin. This value is greatly increased during growth in low concentrations of streptomycin and neomycin, antibiotics which are known to stimulate misreading in vitro. Of the specific types of misreading which streptomycin stimulates in vitro, only misreading of the CGU and CGC arginine codons could give rise to illegitimate incorporation of cysteine. In agreement, partial arginine starvation increases the incorporation of 35S-cysteine into flagellin in a relA- mutant, with or without streptomycin, but has no such effect in its isogenic relA+ partner- Assuming from these results that 35S-cysteine incorporation into flagellin reflects misreading of CGU/C coda, we deduce a misreading probability per codon in the range of 10(-4).

Errors of heterologous protein expression.

Missense substitutions and processivity errors in the translation of heterologous proteins are expected to occur at higher frequencies than the corresponding errors of normal translation. The resulting error-containing products may overload chaperone systems. Likewise, there may be a risk of an immunogenic response to heterologous proteins introduced into vertebrates. Recent work has been carried out on the mechanisms by which such errors arise and on their occurrence in cloned, heterologous gene products.

spoT, a new genetic locus involved in the stringent response in E. coli

Abstract A new genetic locus, spo T, whose product is involved in the stringent response to amino acid starvation, maps very close to 72 min on the E. coli chromosome. The locus is defined by a spontaneous mutant allelle, spo T-, whose phenotypic effects are: virtually no pppGpp is produced during amino acid starvation; ppGpp is overproduced; and the stability of ppGpp upon reversal of the stringent response is greatly increased. All three phenotypic effects are recessive to wild type in heterozygotes. These three phenotypic effects are best accounted for by postulating that the spo T gene product plays a role in the phosphorylation of ppGpp to pppGpp preparatory to further metabolism. Since the stability of ppGpp has also shown to increase following a carbon and energy source downshift (Gallant, Margason, and Finch, J. Biol. Chem. 247 : 6055), we suggest that the activity of the spo T gene product is regulated by some consequence of downshift.

Control of RNA synthesis in Escherichia coli: I. Amino acid dependence of the synthesis of the substrates of RNA polymerase☆

Abstract A study of the amino acid dependence of ribonucleoside triphosphate synthesis and its relationship to the amino acid dependence of RNA synthesis in Escherichia coli has led to the following findings: 1. (1) The conversion of UMP to UTP and CTP by plasmolyzed cells is partially amino acid-dependent in two unrelated stringent strains, but is amino acid-independent in a relaxed mutant derived from one of them. 2. (2) In whole cells of a stringent strain, the levels of UTP and CTP shrink abruptly upon exhaustion of a limiting supply of a required amino acid, at the same time as the rate of RNA synthesis is reduced. 3. (3) In whole cells of a stringent strain, the incorporation of 32 PO 4 into all four ribonucleoside triphosphates is amino acid-dependent; incorporation into ATP shows the most severe amino acid-dependence. 4. (4) The amino acid-dependence of ribonucleoside triphosphate generation in stringent strains cannot be an indirect consequence of the amino acid-dependence of RNA synthesis, because RNA synthesis can be inhibited in any of three different ways without inhibiting ribonucleoside triphosphate generation. RNA synthesis was inhibited at the RNA polymerase level with actinomycin D or proflavine without inhibiting the synthesis of UTP or CTP. RNA synthesis could also be prevented by uracil starvation without significant inhibition of ATP or GTP synthesis. We suggest that the RC control system responds to amino acid deficiency by limiting ribonucleoside triphosphate synthesis, which in turn makes RNA synthesis amino acid-dependent.

Dual function of the λ prophage repressor

Abstract Kinetic experiments with lysogens of a cI temperature-sensitive mutant of phage λ have shown that there are at least two sequential functions under direct cI control. The expression of the first of these—which cannot occur if protein synthesis is inhibited—leads to detachment of the prophage from the bacterial chromosome. The second commits the cell to the production of phage even after repression has been restored at low temperature. The two functions can also be distinguished by their different nutritional requirements: the first function can occur in the absence of thymine, while the second cannot.

An estimate of the global error frequency in translation

SummaryElectrophoretic heterogeneity in a set of selected proteins is used to estimate the average error frequency during translation. Estimates based upon streptomycin-induced heterogeneity as well as mistranslation of an ochre codon yield an average error frequency of 4x10-4 for normally growing cells.

Anomalous synthesis of ppGpp in growing cells

In E. coli cells, accumulation of ppGpp is normally triggered by conditions that restrict the aminoacylation of tRNA or interfere with carbon/energy source metabolism; in both cases, the nucleotides accumulation is associated with control of stable RNA synthesis and is generally believed to bring it about. We have found an anomalous situation wherein vigorously growing cells accumulate a high level of ppGpp and there is no restriction of stable RNA synthesis. This occurs when wild-type cells are shifted up from an abnormally low growth temperature to one in the optimal range (35 degrees C-40 degrees C). The effect is partly, but not entirely, dependent upon the presence of a functional relA gene product. These results appear to call into question the simpler interpretations of the role of ppGpp in the control of stable RNA synthesis.

Error propagation in viable cells

Error propagation is the process, predicted by theoretical models, whereby errors in translating the genetic code will beget fresh errors in successive generations. It has been postulated that error propagation may underly the mortality of cells which display clonal senescence. We have demonstrated the occurrence of error propagation in viable cells of E. coli during growth in a low concentration of streptomycin, a drug which promotes ribosomal ambiguity. We monitored error propagation by measuring mistranslation of a specific UAA codon, and measured viability by direct enumeration of both live and dead cells through a sensitive microscopic technique. We find that the error frequency may be artificially increased by at least an order of magnitude without generating any detectable increase in the proportion of dead cells or of cells whose descendents are doomed to clonal senescence. The error frequency increases gradually over the course of a few generations, in qualitative agreement with the notion of error propagation, and eventually stabilizes at a constant value much higher than normal. The kinetics of this increase agree quantitatively with the Hoffman-Kirkwood and Holliday formulation of error propagation, for parameter values which dictate convergence to a stable error frequency. This convergent behaviour, under conditions of enhanced mistranslation, demonstrates that the normal parameters are well removed from the region of instability in error propagation; even an order of magnitude increase in mistranslation does not tip the translation system into the unstable mode which has been postulated to underly cell senescence. Thus, the error catastrophe theory of cell senescence cannot apply to the translation system of bacteria. We have reviewed experimental data on the fidelity of translation in somatic cells of higher organisms which militate against the notion that the translation system in these cell types could be much closer to the region of instability than in bacteria. These considerations controvert the error catastrophe theory of cell senescence.

On the accuracy of protein synthesis in Drosophila melanogaster

We have investigated the accuracy of protein synthesis in somatic tissue of young and senescent Drosophila melanogaster by means of two-dimensional electrophoresis. Electrophoretic heterogeneity characteristic of translational error could not be detected at either age, placing the error frequency below about 4 X 10(-4) per codon. As a positive control on the efficacy of the technique, we showed that electrophoretic heterogeneity is detectable in bacterial cells grown in streptomycin, and that the error frequency calculated from these data is in good agreement with independent measurements. Such cells can tolerate an error frequency of about 3 x 10(-3) without cell lethality, or error catastrophe. Since the Drosophila somatic error frequency is at least eight times lower, even in senescent organisms, translational error is unlikely to be a cause of senescence in Drosophila.

A novel nucleotide implicated in the response of E. coli to energy source downshift

When E. coli cells are subjected to energy source downshift, the accumulation of RNA (and overall cell growth) is drastically restricted within 1 to 2 min. However, the identity of the primary metabolic signal for this adjustment is a mystery. Earlier studies, and further evidence presented here, show that there is no satisfactory correlation between the sudden adjustment of RNA accumulation and the kinetics of changes in the levels of prospective signalling compounds, such as glycolytic intermediates, ppGpp, ATP, or the three adenylate nucleotides. We have discovered an unusual nucleotide, which we call the phantom spot, whose level decreases dramatically within a minute of downshift, correlating well with the adjustment of RNA accumulation. Preliminary characterization of the phantom spot indicates that it is a triphosphate derived from the guanylate pathway, and suggests that it is a form of GTP with a modification of the imidazole portion of the purine ring. We postulate that this nucleotide serves as a regulatory facsimile of ATP, linking the rate of RNA accumulation and other anabolic processes to the overall rate of phosphorylation.