Kinetics of RNA Polymerase β Subunit Synthesis and Acid End Product Transport in Escherichia coli

Citation

Axe, Douglas D. (1990) Kinetics of RNA Polymerase β Subunit Synthesis and Acid End Product Transport in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ejy5-sm51. https://resolver.caltech.edu/CaltechETD:etd-02222007-113422

Abstract

An approach to modeling the regulation of synthesis of crucial bacterial proteins has been developed. The unique features of this approach are that it focuses on maintenance of a steady state rather than on transitions between steady states, and that stochastic fluctuations in the number of transcripts per cell are treated as the perturbation. It has been used to investigate various models of translational regulation of RNA polymerase β subunit synthesis. The simplest autogenous regulatory mechanism, binding of a single RNA polymerase molecule to the rpoBC mRNA, appears to provide inadequate control. A more sophisticated mechanism, sequential binding of multiple polymerase molecules in a cooperative manner, was shown to dramatically improve the control characteristics.

The interaction between RNA polymerase and rpoBC mRNA was examined experimentally. RNA polymerase was incubated with RNA that is identical to a small portion of the native rpoBC message. Gel mobility-shift assays were performed to detect complexes. The relative amount of RNA in different complexes was determined by radiolabeling the transcript. Data obtained in this way indicate that cooperative binding is occuring.

³¹P NMR studies of intact E. coli cells suggested a difference in membrane function between a plasmid-containing strain and the plasmid-free host. Similar ³¹P NMR experiments were complemented with ¹³C NMR experiments to examine the transport of lactate and acetate through the cytoplasmic membrane during anaerobic glycolysis. Methods were developed to measure cytoplasmic and extracytoplasmic solution volumes and intra- and extracellular acid concentrations using ¹³C NMR. The results demonstrated significant differences in the transport of the two acids. Acetate was determined to permeate the membrane at comparable rates in the dissociated and undissociated forms. The mode of lactate transport in cells that are actively glycolyzing was found to be different from that of cells that have exhausted their supply of glucose. Lactate thus appears to be transported by a system that is sensitive to some indicator of glycolytic activity. It also appears to diffuse across the membrane in both forms.

A kinetic approach was used to deduce constraints on the unidirectional fluxes for a general protein-mediated, ATP-independent transport process. The conclusion was that under certain circumstances, the Ussing-Teorell flux ratio equation applies to protein-mediated transport. From the analysis, an expression was derived for the driving force of such a transport process, and the relationship between this and the net flux is discussed.

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