Analysis of RNA polymerase-promoter complex formation

doi:10.1016/j.ymeth.2008.10.018

Methods

Volume 47, Issue 1, January 2009, Pages 13-24

https://doi.org/10.1016/j.ymeth.2008.10.018 Get rights and content

Abstract

Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus recognition element sequences, in their activities, and in their utilization of transcription factors, and multiple approaches often must be used to provide a framework for understanding promoter regulation. Characterization of RNA polymerase-promoter complex formation in the absence of additional regulatory factors (basal promoter function) can provide a basis for understanding the steps in transcription initiation that are ultimately targeted by nutritional or environmental factors. Promoters can be localized using genetic approaches in vivo, but the detailed properties of the RNAP-promoter complex are studied most productively in vitro. We first describe approaches for identification of bacterial promoters and transcription start sites in vivo, including promoter-reporter fusions and primer-extension. We then describe a number of methods for characterization of RNAP-promoter complexes in vitro, including in vitro transcription, gel mobility shift assays, footprinting, and filter binding. Utilization of these methods can result in determination of not only basal promoter strength but also the rates of transcription initiation complex formation and decay.

Introduction

Bacterial transcription is carried out by a multisubunit RNA polymerase holoenzyme, Eσ, comprised of the core enzyme E (subunits α₂, β, β′ and ω), and one of several σ specificity subunits (a major “housekeeping” sigma factor, such as Escherichia coli σ⁷⁰, or one of a variable number of alternative sigma factors present in different bacterial species) [1], [2], [3], [4]. While the response of RNAP to specialized DNA-binding transcription factors plays a critical role in determining the cell’s transcription program under different growth conditions, the promoter sequences with which RNAP interacts are variable and are a major determinant of the wide range of strength and regulation of gene expression. RNAP-promoter complex formation and transcription initiation in the absence of trans-acting factors are referred to as basal promoter function. In this chapter we focus on methods for observing and studying basal promoter function as a key component of the overall mechanism of transcription regulation. The mechanism of action of regulatory factors (activators or repressors) must ultimately be understood in terms of how they alter the interactions of RNAP with a particular promoter.

An RNAP-promoter complex capable of transcription initiation (an open complex; RP_O) is formed through interactions that involve several regions of RNAP and that span 70–80 bp of promoter DNA (∼−60 to +20 with respect to the transcription start site, +1). Each RNAP subunit, with the exception of ω, participates in these interactions, although a majority of the sequence-specific interactions occur with the σ subunit. In an E. coli Eσ⁷⁰-promoter complex, sequence-specific interactions with σ⁷⁰ occur at the −10 element (with σ⁷⁰ regions 2.3–2.4), the extended −10 element (σ⁷⁰ region 3.0), the −35 element (σ⁷⁰ region 4.2), and the discriminator element immediately downstream of the −10 hexamer (σ⁷⁰ region 1.2) (Fig. 1; [1], [5]). In addition, the C-terminal domain of α subunit can interact sequence-specifically with the UP element, located upstream of the −35 hexamer [6]. Sequence non-specific DNA contacts are mediated by several regions of the large β and β′ subunits in the active site channel and downstream of the start site (as observed in elongation complexes; [7], [8]) and in the spacer region between the −10 and −35 hexamers [9].

Promoters vary widely in overall strength (∼4 orders of magnitude) and in the extent of similarity to the consensus sequences for the recognition elements [1], [10], [11]. The −10 element is the most highly conserved of the promoter elements, but similarities to consensus usually are found in one or more of the other recognition elements as well [6], [12], [13]. The contribution of each recognition element to promoter function depends upon the context in which it is found.

In this review we discuss several methods for investigating RNAP-promoter complex formation, including preliminary identification of the promoter itself using in vivo methods, followed by the characterization of properties of complex formation with RNAP in vitro. Formation of the RNAP-promoter complex can be strongly affected by several parameters in vitro, and effects of these conditions vary with different promoter sequences. These parameters will be discussed as well.

Section snippets

Promoter identification and activity determination in vivo

While bioinformatic analyses can predict some promoters correctly, definitive identification of promoters from sequence information alone remains difficult [14], [15]. In addition, while in vitro methods that examine RNA polymerase-promoter complex formation can be very informative, they can identify interactions that do not correspond to promoters that function in vivo (e.g., “tight-binding sites”, or end-binding sites; [16], [17]). Thus, correct identification of a promoter is established

RNAP-promoter complex formation in vitro

The interaction of purified RNA polymerase with a promoter to form a complex in vitro can be detected and studied by several methods, including in vitro transcription, gel mobility shift (EMSA), footprinting, or filter binding assays. The process of RNAP-promoter complex formation is affected greatly by several parameters, including salt concentration, temperature, template topology, and RNA polymerase concentration. The optimum conditions for RNAP complex formation with a particular promoter

Acknowledgments

We thank Pete Chandrangsu and Justin Lemke for Fig. 2, Fig. 3, and members of our laboratory for helpful discussions. This work was supported by National Institutes of Health Grant R37 GM37048 to R.L.G.

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Review ArticleAnalysis of RNA polymerase-promoter complex formation

Abstract

Introduction

Section snippets

Promoter identification and activity determination in vivo

RNAP-promoter complex formation in vitro

Acknowledgments

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Review Article
Analysis of RNA polymerase-promoter complex formation