RNA Polymerases as Molecular Motors

On the Road

Robert Landick, Terence Strick, Jue Wang, Robert Landick, Terence Strick, Jue Wang

• Publisher: Royal Society of Chemistry
• Year: 2021
• Print ISBN: 9781788013659
• eBook ISBN: 9781839160660
• Edition: 2
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

To thrive, every living cell must continuously gauge and respond to changes in its environment. These changes are ultimately implemented by modulating gene expression, a process that relies on transcription by Nature's most multivalent molecular machine, the RNA polymerase. This book covers progress made over the past decade understanding how this machine functions to compute the cellular state, from the atomistic structural level responsible for chemistry to the integrative level at which RNA polymerase interacts with the other key molecular machineries of the cell.

CHAPTER 1

The Transition from Transcription Initiation to Transcription Elongation: Start-site Selection, Initial Transcription, and Promoter Escape

JARED T. WINKELMAN^a, ^b, BRYCE E. NICKELS^*b AND RICHARD H. EBRIGHT^*a

^a Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA, ^b Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA,
^*E-mail: [email protected], [email protected]

In transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds a turn of promoter DNA to yield an RNAP–promoter open complex containing an unwound “transcription bubble,” and selects a transcription start site (TSS). In the next step of initiation, termed “initial transcription,” RNAP remains bound to the promoter and synthesizes an RNA product of a threshold length of approximately 11–15 nucleotides. In the final step of initiation, termed “promoter escape,” RNAP breaks free of the promoter to yield a transcription elongation complex that synthesizes the rest of the RNA product. As a result of research over the last two decades, we now have a detailed mechanistic understanding of TSS selection, and we now understand broad outlines of initial transcription and promoter escape. Here we review the current understanding of TSS selection, initial transcription, and promoter escape, focusing on these processes as they occur in the best characterized example, transcription initiation by Escherichia coli RNAP-σ⁷⁰ holoenzyme, but also summarizing these processes as they occur in eukaryotic RNAP I, II, and III.

1.1 Introduction

In transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds a turn of promoter DNA to yield an RNAP–promoter open complex containing an unwound “transcription bubble,” and selects a transcription start site (TSS). In the next step of initiation, termed “initial transcription,” RNAP remains bound to the promoter and synthesizes an RNA product of a threshold length of approximately 11–15 nucleotides (nt). In the final step of initiation, termed “promoter escape,” RNAP breaks free of the promoter to yield a transcription elongation complex that synthesizes the rest of the RNA product. As a result of research over the last two decades, we now have a detailed mechanistic understanding of TSS selection, and we now understand the broad outlines of initial transcription and promoter escape. Here we review the current understanding of TSS selection, initial transcription, and promoter escape, focusing on these processes as they occur in the best characterized example, transcription initiation by Escherichia coli RNAP-σ⁷⁰ holoenzyme, but also summarizing these processes as they occur in eukaryotic RNAP I, II, and III.

1.2 Transcription Start-Site (TSS) Selection

1.2.1 Mechanism

In contrast to DNA polymerases, RNAP can initiate nucleic acid synthesis using both primer-independent and primer-dependent mechanisms.^1,7 In primer-independent transcription initiation, RNAP uses an initiating nucleoside triphosphate (NTP) and an extending NTP or, alternatively, uses a non-canonical initiating nucleotide^8,9 [NCIN; an NTP-related compound, such as nicotinamide adenine dinucleotide (NAD⁺) or its reduced form (NADH)] and an extending NTP. In primer-dependent transcription initiation, RNAP uses a short, 2 to ∼5 nucleotide, RNA primer (“nanoRNA”) and an extending NTP.^10,13

In both primer-independent and primer-dependent transcription initiation, TSS selection entails two steps: (1) placing the start-site nucleotide (position +1) and the next nucleotide (position +2) of the template DNA strand into the RNAP active-center product site (“P site”) and addition site (“A site”), respectively; and (2) placing the initiating entity—the initiating NTP or NCIN in primer-independent initiation, or the 3′ nucleotide of the RNA primer in primer-dependent initiation—in the P site and the extending NTP in the A site, respectively.

The position of the TSS relative to promoter core elements (promoter −35 element and −10 element for E. coli RNAP-σ⁷⁰ holoenzyme) is variable.^14,23 The results of a comprehensive analysis of TSS selection by RNAP-σ⁷⁰ holoenzyme indicate that TSS selection most frequently entails placing the template-strand position located 7 nucleotides downstream of the promoter −10 element into the RNAP active-center P site to serve as position +1 ^14,22,24 (see Figure 1.1). However, the TSS can vary over a 5 bp window, allowing placement of template-strand positions located 6, 7, 8, 9, or 10 nucleotides downstream of the promoter −10 element into the RNAP active-center P site to serve as position +1.^14,22,24

Figure 1.1Mechanism of transcription start site (TSS) selection. Changes in TSS selection result from changes in DNA scrunching. The left column shows the TSS positions expressed in terms of distance downstream of the promoter −10 element (6, 7, 8, 9, or 10 nt downstream). The centre column shows the structures of anti-scrunched (TSS = 6), unscrunched (TSS = 7), and scrunched (TSS = 8, 9, 10) RNAP–promoter open complexes. Anti-scrunching is indicated by stretched lines between nucleotides; scrunching is indicated by bulged-out nucleotides. Positions of scrunched and anti-scrunched nucleotides are speculative. Gray, RNAP; yellow, σ; blue, −10-element nucleotides; purple, discriminator nucleotides; P and A, RNAP active center NTP-binding sites; boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides; nucleotides downstream of the −10 element are numbered). The right column shows the observed percentage of transcription start sites at each position as measured by Vvedenskaya et al. ¹⁴

The variability in TSS selection raises the structural question of how placement of the template strand relative to the RNAP active center can vary by up to 5 nucleotides, which corresponds to variation by up to at least ∼17 Å (5 nucleotides × ∼3.4 Å per nucleotide). In principle, this variability could be accommodated by differences in RNAP conformation, differences in DNA conformation, or both.

Results from a series of experiments have now revealed, definitively, that variability in placement of the template strand relative to the RNAP active center is mediated by differences in DNA conformation, specifically differences in the extent of transcription-bubble unwinding^22,23,25,26 (see Figure 1.1). At most promoters, the energetically most favorable configuration of the RNAP–promoter open complex is one that contains an unwound transcription bubble 13 nucleotides in length and that places the template-strand position 7 nucleotides downstream from the promoter −10 element in the RNAP active-center P site. In order for TSS selection to occur at positions 8, 9, or 10 nucleotides downstream of the promoter −10 element, the downstream DNA duplex is further unwound by an additional 1, 2, or 3 bp, respectively; the unwound DNA is pulled into and past the RNAP active center, and the unwound DNA is accommodated as single-stranded DNA bulges within the transcription bubble, yielding a “scrunched” complex (TSS = 8, 9, or 10 in Figure 1.1). In order for TSS selection to occur 6 nucleotides downstream of the promoter −10 element, the opposite occurs: downstream DNA is rewound by 1 bp, downstream DNA is extruded from the RNAP active center by 1 bp, and the extrusion of DNA from the RNAP active center is accommodated by stretching DNA within the unwound transcription bubble, yielding an “anti-scrunched” complex (TSS = 6 in Figure 1.1).

Scrunching and anti-scrunching during TSS selection have two defining, experimentally detectable, hallmarks: (1) as the position of the TSS changes, the position of the RNAP leading edge relative to DNA changes, but the position of the RNAP trailing edge relative to DNA does not c...

Table of contents

• Cover
• Chemical Biology
• Title
• Copyright
• Preface to the Second Edition
• Foreword to the First Edition
• Preface to the First Edition
• Contents
• Structural Atlas
• Chapter 1 The Transition from Transcription Initiation to Transcription Elongation: Start-site Selection, Initial Transcription, and Promoter Escape
• Chapter 2 Molecular Basis for Transcriptional Fidelity Control by RNA Polymerase II
• Chapter 3 RNA Polymerase as a Torsional Motor
• Chapter 4 RNA Polymerase-associated Transcription Elongation Factors
• Chapter 5 Rho Termination Factor: One Ring to Bind Them All
• Chapter 6 Global Regulation of Transcription by Nucleotides and (p)ppGpp
• Chapter 7 Helicases and the Obstructive RNAP
• Chapter 8 Transcription–Replication Conflicts, Resolution and Coregulation
• Chapter 9 Using Single-cell RNA Measurements to Decipher the Stochastic Kinetics of Transcription
• Chapter 10 Molecular Dynamics Simulations and Kinetic Network Models Elucidate RNA Polymerase Transcription Elongation Mechanisms
• Chapter 11 Epilogue
• Subject Index