Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. II. Fraction of RNA polymerase engaged in the synthesis of stable RNA at different steady-state growth rates

The relative rates of stable RNA synthesis (rate of stable synthesis/rate of total RNA synthesis) were determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36 doublings/h) and glucose/amino acids (μ = 2.10 doublings/h) media. The relative rates were 0.29, 0.50 and 0.66 at these growth rates. From the relative rates, the fraction of RNA polymerase engaged in the synthesis of stable RNA, ψ_s, was calculated to be 0.22, 0.36 and 0.48, respectively, by taking into account the difference between the RNA chain growth rate of stable and that of unstable RNA. The relationship between these ψ_s values and μ and our previously determined chain growth rate of stable RNA has two implications for the control of RNA synthesis during a nutritional shift-up: (1) the increase in the net rate of RNA synthesis after a shift-up results from a transfer of RNA polymerase molecules from unstable to stable RNA genes, and a concomitant increase in the stable RNA chain growth rate, but does not require an activation of RNA polymerase; (2) the synthesis of functioning RNA polymerase enzymes is subject to a growth rate-dependent control.     

Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates

The levels of initiation factors and other translational components were compared in crude lysates of Escherichia coli grown at different rates. Cells were grown in media containing [35S]sulfate and different carbon sources, and harvested during mid-exponential phase after about 10 generations of balanced growth. Initiation factors (IF), elongation factors (EF), and a number of ribosomal proteins were identified unambiguously in gel patterns following two-dimensional polyacrylamide gel electrophoresis. The molar concentration of each protein was calculated from measurements of the radioactivity in excised gel spots and knowledge of the sulfur content of each protein. We found that the ribosomal proteins and elongation factors were present in equimolar amounts except for L7/L12 and EF-Tu which were 4-fold and 5-fold more abundant, respectively, and that the levels of each protein increased in proportion to growth rate. These results are similar to ones obtained previously by other methods, and serve to confirm the validity of our method. We found that the levels of IF2a and IF3 also were approximately proportional to growth rate. We also measured the levels of all three initiation factors using a radioimmune assay, showed that the factors are present in equimolar amounts, and confirmed that their abundance increased in parallel with ribosomes. We conclude that initiation factor levels are coordinately regulated with one another and with ribosome and elongation factor levels.     

The inefficiency of ribosomes functioning in Escherichia coli growing at moderate rates

It is generally agreed that ribosomes function with reduced efficiency (i.e. a smaller proportion is actually engaged in protein synthesis) in bacteria growing at low growth rates (doubling times greater than 2 h). This paper examines whether the efficiency is constant in bacteria growing at various rates corresponding to doubling times of less than 2 h. Because isotopic methods cannot be used in very rich media, turbidimetric methods have been extended to follow the kinetics of growth immediately following the shift-up of cultures of Escherichia coli ML308 growing in glucose minimal medium or succinate minimal medium into a very rich medium supporting a balanced doubling time of 17.4 min. It is concluded that the efficiency of ribosome participation in protein synthesis is higher in the very rich medium than in the two minimal media, which support doubling times of 43 and 65 min, respectively, at 37 degrees C.     

Arabinose-leucine deletion mutants of Escherichia coli B-r

The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.