Single-molecule studies of RNAPII elongation

Abstract

Elongation, the transcriptional phase in which RNA polymerase (RNAP) moves processively along a DNA template, occurs via a fundamental enzymatic mechanism that is thought to be universally conserved among multi-subunit polymerases in all kingdoms of life. Beyond this basic mechanism, a multitude of processes are integrated into transcript elongation, among them fidelity control, gene regulatory interactions involving elongation factors, RNA splicing or processing factors, and regulatory mechanisms associated with chromatin structure. Many kinetic and molecular details of the mechanism of the nucleotide addition cycle and its regulation, however, remain elusive and generate continued interest and even controversy. Recently, single-molecule approaches have emerged as powerful tools for the study of transcription in eukaryotic organisms. Here, we review recent progress and discuss some of the unresolved questions and ongoing debates, while anticipating future developments in the field. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

Introduction

Much of our current understanding of transcription has come from ensemble-based measurements, performed using traditional biochemical approaches in which RNA polymerases and their accessory factors were first isolated, and then their behavior analyzed. These careful measurements have been complemented by structural studies that provide breathtaking ‘still pictures’ of the transcriptional apparatus [54], [100] with spatial resolution down to the atomic-scale. The information from such structures has been integrated to the point that we can now construct ‘movies’ of the nucleotide addition cycle [11] based on snapshots of RNAPII in different intermediate states. However, despite these successes, our understanding of the detailed structural rearrangements and the kinetics of the steps connecting these states is far from complete.

In particular, every substep of the nucleotide addition cycle is a potential target of an intricate network of gene regulatory pathways, and every nucleotide addition cycle is subject to scrutiny by fidelity control (error correction) mechanisms that ensure faithful transcription of the genetic code. Individual enzymes frequently switch between periods of productive elongation and off-pathway states with regulatory significance, often in an apparently stochastic manner. It is notoriously difficult to study such complex pathways in bulk biochemical experiments, where it is impossible or impractical to resolve rare stochastic events, and mixed populations of enzymes in both active and inactive states co-exist. Single-molecule approaches, by contrast, can avoid the complications of ensemble averaging, providing dynamic information about the kinetics of individual enzymes with comparatively high spatial and temporal resolution.

Over the past two decades, with the ability to probe directly the transient, unsynchronized behavior of RNAP molecules, single-molecule techniques have provided fresh insights into the mechanisms of transcript elongation. Broadly speaking, three classes of single-molecule techniques have been applied to the study of transcription: single-molecule fluorescence (e.g., single-molecule Förster resonance energy transfer, or smFRET), atomic force microscopy (AFM), and displacement-based methods that attach tiny particles to polymerases and track their motions, e.g., optical traps, magnetic tweezers, and tethered particle motion (TPM) assays (reviewed in [37]). In particular, single-molecule optical trapping assays of transcription have progressed dramatically, tracking the motion of RNA polymerase along DNA with a spatial resolution down to single base-pairs [1]. Past work has largely concentrated on the prokaryotic form of RNAP [41], which is straightforward to initiate on a DNA template from a promoter site, after which elongation can be studied. Only recently has it become practical to circumvent the complex initiation process generally required for eukaryotic RNA polymerases, which involves multiple transcription initiation factors. In vitro, functional elongation complexes can be created in the absence of such factors by assembling a scaffold of DNA and RNA around the enzyme that mimics the transcription bubble assembly. Using this approach, high-resolution optical trapping studies have since been employed to study the mechanochemistry of RNAPII [31], [67], as well as to probe its interactions with factor TFIIS [31], and to study transcription through nucleosomes [44]. Complementary work using FRET-based approaches has begun to unravel the interactions of RNA polymerase with nucleic acids at nanometer spatial resolution [2], [3], [15], [72].

In parallel with these developments, dramatic progress has been made using live-cell imaging approaches, based on the expression in vivo of fluorescent proteins. Single-molecule imaging studies of transcription in vivo have become feasible as well, and have led to a picture of gene expression in which many key molecular players (transcription factors, mRNA transcripts, etc.) exist at the level of only a few molecules per cell. As a consequence of the small numbers, stochastic events in transcriptional and translational processes become important in determining cellular fates [16]. Recently, single-molecule studies in vivo have been extended to the study of eukaryotic transcription as well, visualizing the kinetics of the initiation and elongation phases of transcription, revealing heterogeneities in the rates of transcription at different stages of the cell cycle [66].

Here, we review recent progress in single-molecule assays developed to study eukaryotic transcriptional elongation, the kinetics and fidelity control of the nucleotide addition cycle, and the interactions of RNAPII with nucleosomes and transcription factors. We end by presenting opportunities and challenges for future single-molecule studies, and discuss their potential synergy with emerging, genome-wide approaches to eukaryotic transcription.