Single-Molecule Studies of Transcription: From One RNA Polymerase at a Time to the Gene Expression Profile of a Cell

Abstract

Single-molecule techniques have emerged as powerful tools for deciphering mechanistic details of transcription and have yielded discoveries that would otherwise have been impossible to make through the use of more traditional biochemical and/or biophysical techniques. Here, we provide a brief overview of single-molecule techniques most commonly used for studying RNA polymerase and transcription. We then present specific examples of single-molecule studies that have contributed to our understanding of key mechanistic details for each different stage of the transcription cycle. Finally, we discuss emerging single-molecule approaches and future directions, including efforts to study transcription at the single-molecule level in living cells.

Introduction

Transcription is the key step of gene expression and regulation in which the information encoded in genomic DNA is transcribed into RNA. The products of transcription can be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), or other types of RNA molecules such as ribozymes. In addition, many of these different RNA molecules are processed to generate an increasingly diverse array of small RNA species which themselves can influence gene expression.1, 2, 3 Numerous cis- and trans-acting factors work together to establish a complex network of regulatory features, allowing precise control over the expression of any given gene.4, 5, 6 This regulation is achieved through the combined effects of promoter DNA sequences that dictate the sites of transcript initiation, along with the effects of a multitude of transcription factors and other regulatory elements that can influence the efficiency of transcript initiation, elongation, and/or termination. In eukaryotes, transcription regulation is further complicated by the higher-order organization of chromatin structure, with the positioning of nucleosomes and establishment of repressive chromatin structures greatly influencing the organization and regulation of gene expression.6, 7, 8, 9 At the heart of this regulatory network lies RNA polymerase (RNAP), which is the protein machinery directly responsible for RNA synthesis. The simplest RNAPs come from bacteriophages and consist of single polypeptides capable of carrying out all the basic steps of transcription.¹⁰ In prokaryotes, a single multisubunit RNAP is responsible for all RNA production, whereas in eukaryotes many different types of RNAP [e.g., polymerase I (Pol I), II, III] divide the labor required for production of different classes of RNA molecules.11, 12, 13

Before transcription can begin, RNAP must locate the promoter sequence that lies upstream from the gene that is to be expressed in a process that can be referred to as the promoter search. Following the promoter search, transcription itself can be divided into three stages: initiation, elongation, and termination; and all RNAPs, regardless of their origin, must complete the same basic set of reactions in order to generate RNA transcripts. The transcriptional machinery first unwinds the template DNA before beginning RNA synthesis, and this process is referred to as open complex formation. Bacteriophage and prokaryotic RNAP holoenzymes can conduct all of these steps with no need for accessory factors, whereas eukaryotes and archaea require a large preinitiation complex composed of RNAP and several additional transcription factors.14, 15, 16 After open complex formation, RNAP undergoes abortive initiation, wherein it synthesizes many short transcripts ∼ 9–11 nt in length, until it finally escapes the promoter and begins elongating the RNA.17, 18, 19 During elongation, RNAP translocates along the template DNA while catalyzing successive addition of ribonucleotides [ribonucleotide triphosphates (rNTPs)] to the growing RNA chain. RNAP is highly processive during elongation, but its forward motion is not monotonic; rather, it exhibits frequent pauses and backtracking,20, 21 which are often coupled to proofreading mechanisms necessary to ensure fidelity.21, 22 Once a gene is transcribed, transcription must be terminated. Escherichia coli RNAP terminates transcription through two distinct mechanisms, either intrinsic termination or rho-dependent termination.23, 24 During intrinsic termination, a hairpin formed in the nascent RNA destabilizes the elongation complex (EC). In rho-dependent termination, rho disrupts transcribing RNAP through a process coupled to ATP-dependent translocation along the nascent transcript. Termination in eukaryotes is less well understood and is coupled to 3′ end processing of the transcript; however, the underlying processes may share some mechanistic similarities with E. coli RNAP. It has been proposed that either a polyadenylation sequence in the RNA changes the factors associated with the polymerase, making it less processive, or some “rho-like” helicase may bind the 5′ RNA end generated by the cleavage at the polyadenylation sequence.²⁵

RNAP is the most important component of the transcription apparatus, and much of the existing knowledge regarding the mechanisms by which it functions have been garnered from single-molecule studies. The power of these studies lies in their ability to observe and measure individual molecules in real time, thereby eliminating the need for ensemble averaging and allowing direct detection of rare or transient intermediates within heterogeneous populations of molecules. In addition, some single-molecule techniques can physically manipulate individual molecules of RNAP, offering the ability to study the response of RNAP to externally applied forces as well as measure the forces that RNAP is able to exert. In this review, we highlight a number of critical single-molecule transcription studies, with emphasis placed on the latest progress in the field as well as future avenues of research that will push forward our understanding of transcription.