Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms

Abstract

Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ⁷⁰. The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription.

Graphical abstract

Fig. 1

A) Structure of bacterial RNAP core enzyme (left) and holoenzyme (right); B) Schematic of a bacterial promoter showing consensus − 10 and − 35 elements, extended − 10 and discriminator elements and their interaction with different modules of σ⁷⁰.

Fig. 2

A) Schematic describing intermediates in open complex pathway showing status of promoter melting, heparin sensitivity and relative kinetic profiles of individual steps. B) schematic showing models for promoter melting and active centre cleft loading of DNA: clamp opening model (top), couple melt and load model (middle) and external unwinding model (bottom); in all the models the RNAP clamp is depicted in red (for closed clamp) and green (for open clamp), double stranded DNA is in blue and melted single stranded DNA is in orange; C) Structure of Mtb RNAP-promoter intermediate complex showing a partially melted DNA bubble (left) and an Mtb RNAP open complex with fully melted DNA bubble (right) (Adapted from Boyaci etal, Nature, 2019 ; used with permission).

Fig. 3

A) Schematic of the three proposed models for mechanism of initial transcription: (top), transient excursions; (middle) inchworming; (bottom) scrunching.; B) smFRET study showing scrunching during initial transcription; single molecule assay showing increasing FRET efficiency (decrease in distance) for dyes in positions − 15 (Cy3b) and + 15 (Alexa647) of a lacCONS promoter. Subpanels show E ∗ histograms of open complex (RP_o) and initial transcribing complexes with up to 7-nt RNA (RP_ITC, ≤ 7). The histograms show distributions of free DNA (lower- E ∗ species) and the RNA polymerase (RNAP)–DNA complexes. An increase in FRET efficiency indicates a decrease in distance in going from RP_o to RP_ITC and supports the scrunching model for initial transcription; (from Kapanidis et al, Science 2006 , used with permission). C) magnetic tweezer study showing 1 bp scrunching per nucleotide addition cycle during initial transcription. (top) the end-to-end extension of a mechanically stretched, negatively supercoiled or positively supercoiled single DNA molecule containing a single promoter is monitored. Unwinding of n turn of DNA by RNAP result in the compensatory loss of n negative supercoils or gain of n positive supercoils and a readily detectable movement of the bead; (bottom) magnetic tweezers data showing scrunching of DNA during initial transcription (adapted from Revyakin et al, Science, 2006 ; used with permission).

Fig. 4

A) smFRET assay showing pausing in initial transcription; (top): left, RP_o; right, initial transcribing complex (ITC). Donor is in green; acceptor in red; σ⁷⁰ in orange; RNAP in grey, except for the β subunit (omitted for clarity) and regions protruding from the cut-away plane (in yellow); template strand in blue; non-template strand in teal; nascent RNA in red; and RNAP active site in pink. The penta-His antibody anchors RP_o to the surface. The initial FRET efficiency is low; upon NTP addition, scrunching moves the acceptor closer to the donor, increasing FRET efficiency; (middle): lacCONS DNA fragment for FRET assay; the − 10/−4 pre-melted region is in blue; (bottom): time trace showing an increase to E ∗ ∼ 0.37 upon adding 80 μM UTP and GTP to form RP_ITC≤7. The NTP addition point is marked with a dashed line. Frame time: 20 ms. DD trace (green trace, top), donor emission upon donor excitation; DA trace (red trace, top), acceptor emission upon donor excitation; AA trace (grey trace, top), acceptor emission upon acceptor excitation. DD and DA are used for calculating apparent FRET efficiency E ∗; B) Model for pausing in initial transcription showing the different elements in the RNAP-promoter complex in play; (top): productive path for initial transcription. Coloured columns show translocational registers adopted by growing RNA (in black). Binding site for incoming NTP is in light purple; σ3.2 loop is shown in three putative conformations (in orange). The translocational equilibrium for RP_ITC6 is controlled by several regulatory factors that modulate the lifetime of paused states arising from a pre-translocated RP_ITC6; (middle): abortive path for initial transcription, branching from the pre-translocated RP_ITC6 state of the productive path; (bottom): path for the formation of stable backtracked scrunched states, branching from the pre-translocated RP_ITC6 state of initial transcription during NTP starvation that limits RNA synthesis to 7 nt in length. (adapted from Duchi et al, Mol Cell, 2016 ; used with permission).

Fig. 5

A) (top): Design of single molecule assay for detecting retention of σ⁷⁰ during elongation; (bottom): images of the same microscope field of view of AF488–DNA (blue), Cy5–σ⁷⁰ (red), and transcript-hybridization probe (green) taken at the specified times. B) (top): Two examples of time records of transcript probe (green) and σ⁷⁰ (red) fluorescence, each colocalized at a DNA spot σ⁷⁰ fluorescence departs before the time interval (shaded) during which transcript probe fluorescence is present. (bottom) σ⁷⁰ fluorescence persists throughout transcript probe interval. (C) The fraction (± SEM) of TECs that retain σ⁷⁰ at the time transcript probe is first detected on the TEC (reprinted from Harden et al, PNAS, 2016 ; used with permission).