mRNA composition and control of bacterial gene expression

Abstract

The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of beta-galactosidase expressed from three different promoter-lacZ fusions (P(spc)-lacZ, P(RNAI)-lacZ, and P(RNAII)-lacZ) was measured (i) in a relA(+) background during exponential growth at different rates and (ii) in relA(+) and DeltarelA derivatives of Escherichia coli B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp.

FIG. 1

Growth rate dependence of β-galactosidase expression from P_spc, P_RNAI, and P_RNAII. Strains SL106, YX101, and YX102, respectively, were used, and the growth media (given in order from the lowest to the highest growth rate) were glycerol minimal medium, glucose minimal medium, glucose-amino acids, and LB-glucose (see Materials and Methods).

FIG. 2

β-Galactosidase expression from P_spc during the stringent and the relaxed response. The stringent (relA⁺) strain SL104 (a and c) and the relaxed (ΔrelA) strain SL111 (b and d) were grown exponentially (●) in glucose-amino acids medium. Mild amino acid deprivation was induced in part of the culture (▴) by treatment with 3 μg of pseudomonic acid/ml. (a and b) culture mass density (OD₆₀₀) as a function of time; (c and d) β-galactosidase activity plotted against cell mass density. Values are normalized to the β-galactosidase activity and culture mass (OD₆₀₀) observed at the time of drug addition. τ, doubling time.

FIG. 3

β-Galactosidase expression from P_RNAI and P_RNAII during the stringent and the relaxed response. Stringent and relaxed strains carrying P_RNAI-lacZ and P_RNAII-lacZ operon fusions were grown exponentially in glucose minimal medium and treated with pseudomonic acid (3 μg/ml), as described in the legend for Fig. 2. (a and b) P_RNAI-lacZ fusion strains YX101 (relA⁺) and YX103 (ΔrelA); (c and d) P_RNAII-lacZ fusion strains YX102 (relA⁺) and YX104 (ΔrelA). The culture mass (OD₆₀₀) values and β-galactosidase activities were normalized to the values observed at time zero (addition of pseudomonic acid); only the differential plots, of β-galactosidase activity versus culture mass, are shown.

FIG. 4

Growth rate dependence of the β-galactosidase specific activity expressed from P_spc in strain SL106 and of transcription and translation parameters that determine this specific activity (see the text and equation 1). (a) β-Galactosidase specific activity; (b) relative abundance of lacZ mRNA as a proportion of total mRNA; (c) rate of translation initiation of lacZ mRNA in relative units; (d) average rate of translation initiation of total (bulk) mRNA in translations per minute per average mRNA molecule. This rate can be found either from the total rate of protein synthesis per amount of mRNA [(dP_t/dt)/R_m] or from the peptide chain elongation rate (c_p) and the average distance of ribosomes on the mRNA (d_r [see Table 2, footnote u]). Data represent interpolated values taken from Table 2 (see Table 2, footnotes f, p, h, and u, for the data in panels a, b, c, and d, respectively).