Pease A J, Wolf R E
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
J Bacteriol. 1994 Jan;176(1):115-22. doi: 10.1128/jb.176.1.115-122.1994.
In Escherichia coli K-12 strain W3110, the amount of 6-phosphogluconate dehydrogenase relative to that of total protein, i.e., the specific enzyme activity, increases about threefold during growth in minimal media over the range of growth rates with acetate and glucose as sole carbon sources. Previous work with gnd-lac operon and protein fusion strains indicated that two steps in the expression of the gnd gene are subject to growth rate-dependent control, with at least one step being posttranscriptional. With both Northern (RNA) and slot blot analyses, we found that the amount of gnd mRNA relative to that of total RNA was 2.5-fold higher in cells growing in glucose minimal medium than in cells grown on acetate. Therefore, since the total mRNA fraction of total RNA is essentially independent of the growth rate, the amount of gnd mRNA relative to that of total mRNA increases about 2.5-fold with increasing growth rate. This indicates that most of the growth rate-dependent increase in 6-phosphogluconate dehydrogenase can be accounted for by the growth rate-dependent increase in gnd mRNA level. We measured the decay of gnd mRNA mass in the two growth conditions after blocking transcription initiation with rifampin and found that the stability of gnd mRNA does not change with growth rate. We also used a gnd-lacZ protein fusion to measure the functional mRNA half-life and found that it too is growth rate independent. Thus, the growth rate-dependent increase in the level of gnd mRNA is due to an increase in gnd transcription, and this increase is sufficient to account for the growth rate regulation of the 6-phosphogluconate dehydrogenase level. The dilemma posed by interpretations of the properties of gnd-lac fusion strains and by direct measurement of gnd mRNA level is discussed.
在大肠杆菌K-12菌株W3110中，以乙酸盐和葡萄糖作为唯一碳源时，在整个生长速率范围内，6-磷酸葡萄糖酸脱氢酶相对于总蛋白的量，即比酶活性，在基本培养基中生长期间增加约三倍。先前对gnd-lac操纵子和蛋白质融合菌株的研究表明，gnd基因表达的两个步骤受到生长速率依赖性控制，至少有一个步骤是转录后调控。通过Northern（RNA）分析和狭缝印迹分析，我们发现，在葡萄糖基本培养基中生长的细胞中，gnd mRNA相对于总RNA的量比在乙酸盐上生长的细胞高2.5倍。因此，由于总RNA中的总mRNA部分基本上与生长速率无关，gnd mRNA相对于总mRNA的量随着生长速率的增加而增加约2.5倍。这表明，6-磷酸葡萄糖酸脱氢酶中大部分生长速率依赖性增加可归因于gnd mRNA水平的生长速率依赖性增加。在用利福平阻断转录起始后，我们测量了两种生长条件下gnd mRNA质量的衰减，发现gnd mRNA的稳定性不会随生长速率而变化。我们还使用gnd-lacZ蛋白质融合来测量功能性mRNA半衰期，发现它也与生长速率无关。因此，gnd mRNA水平的生长速率依赖性增加是由于gnd转录增加，并且这种增加足以解释6-磷酸葡萄糖酸脱氢酶水平的生长速率调节。文中讨论了对gnd-lac融合菌株特性的解释以及对gnd mRNA水平直接测量所带来的困境。
