Thomas D. Yager

Escherichia coli σ70 and NusA proteins: II. Physical properties and self-association states☆

Abstract In this paper we examine the physical properties and potential for self-association of the Escherichia coli transcription factors, σ 70 and NusA. We show, by a combination of chemical crosslinking, equilibrium and velocity sedimentation, quasi-elastic light scattering, and small-angle X-ray scattering that NusA exists as a monomer at KCl concentrations between 0.01 and 1.5 m , and that σ 70 exists as a monomer at KCl concentrations between 0.1 and 1.5 m . The shape and hydration characteristics of each of these monomeric proteins are also examined. The results serve as background for the companion paper in which a thermodynamic analysis is made of the interactions of these transcription factors with E. coli core RNA polymerase in solution and as a component of the functional transcription complex.

Thermodynamic analysis of the transcription cycle in E. coli

The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.

7 Quantitative Aspects of the Transcription Cycle in Escherichia coli

OVERVIEW A quantitative analysis of the transcription cycle of E. coli is presented that incorporates both equilibrium and kinetic elements. We consider the various stages of transcription initiation, the properties of the elongation complex, and the relationship of elongation to termination. Thermodynamic descriptions of the transcription cycle can be cast in the form of free energy diagrams. These can be combined with kinetic descriptions based on activation energy barrier diagrams to provide the basis for a quantitative description of regulatory processes at each stage of the cycle. INTRODUCTION In E. coli , control over gene expression is exerted primarily at the level of transcription.3 In this chapter, we deal almost exclusively with the events of transcription catalyzed by the DNA-dependent RNA polymerase of E. coli , since we know far more about this polymerase than about any other. However, it is becoming evident that the RNA polymerases of eukaryotes are related to that of E. coli at both the evolutionary (Sweetser et al. 1987) and the functional levels. We expect that within a few years it will be possible to describe the behavior of eukaryotic transcription systems in comparable detail and that many of the general principles described here will apply to eukaryotes as well. The Transcription Cycle The transcription cycle, by which we mean the series of phases through which the RNA polymerase passes in directing transcript formation, is presented in schematic form in Figure 1. This cycle is conventionally divided into the phases of initiation, elongation, and termination. Each of...

The Time‐Course of Hatching Enzyme Secretion in the Sea Urchin Strongylocentrotus purpuratus

By two independent methods, we have determined approximately the time‐course of hatching enzyme secretion in the sea urchin Strongylocentrotus purpuratus. A quick‐kill method indicates that a significant fraction of the enzyme is secreted between 90% and 97% of the fertilization‐hatching interval. A direct assay method indicates that the remainder of the enzyme is secreted on either side of the 90–97%“window”. The entire period of secretion spans from 75% to 100% or more of the fertilization‐hatching interval. For embryos raised at 15°C this translates to an interval of 4.8 or more hr.