Watching the bacterial RNA polymerase transcription reaction by time-dependent soak-trigger-freeze X-ray crystallography

Abstract

RNA polymerase (RNAP) is the central enzyme of gene expression, which transcribes DNA to RNA. All cellular organisms synthesize RNA with highly conserved multi-subunit DNA-dependent RNAPs, except mitochondrial RNA transcription, which is carried out by a single-subunit RNAP. Over sixty years of extensive research elucidated the structures and functions of cellular RNAPs. In this review, we introduce a brief structural feature of bacterial RNAP, the most well characterized model enzyme, and a principle of novel experimental approach known as “Time-dependent soak-trigger-freeze X-ray crystallography” which can observe RNA synthesis reaction at atomic resolution in real time for elucidating the fundamental mechanism of cellular RNAP transcription.

Transcription – copying genomic DNA to RNA – in all three domains of life, bacteria, archaea and eukaryote, is performed by DNA-dependent RNAP [1–6]. Except single-subunit mitochondrial RNAP, which is related to bacteriophage single-subunit RNAP [7], all other cellular RNAPs are multi-subunit complexes. The overall architecture of multi-subunit RNAPs and the catalytic mechanism of RNA synthesis are highly conserved from bacteria to human [8–14]. In bacteria, the catalytic core enzyme consists of five subunits (α₁, α₂, β, β’, ꞷ) and binding of transcription initiation factor σ [15–18] forms a holoenzyme to synthesize RNA from specific DNA sites called promoters [19, 20] (Figure 1A). The cleft between β and β’ subunits forms a DNA binding main channel for loading a template strand DNA (tDNA) entry into the active site where RNA synthesis takes places via two Mg²⁺ mediated catalysis of nucleotidyl transfer reaction [8, 10–13, 21–23]. Ribonucleoside triphosphates (rNTPs), substrate of RNA synthesis, are delivered to the active site through a funnel shaped secondary channel [24], which also serves as a binding site and access route to the RNAP active site for proteins such as elongation factors GreA/GreB and a transcription regulator DksA [25–27]. After a few rounds of abortive transcription, a nascent RNA and/or DNA scrunching trigger dissociation of the σ factor from the core enzyme for escaping from promoter DNA and RNA continues to extend toward RNA exit channel to form a stable transcription elongation complex. During transcription elongation, transcription factors NusA and NusG associate with RNAP to regulate transcription speed, RNA folding and recuring other regulatory factors and enzymes such as ribosome for the transcription and translation coupling. During the nucleotidyl transfer reaction, RNAP changes its conformation including trigger loop (TL)/trigger helix (TH) and bridge helix (BH). TL/TH undergoes conformational change from a disordered loop (TL) to two α helices (TH) upon binding of a cognate rNTP at the active site, which facilitates the nucleotidyl transfer reaction. The conformational change of TL/TH coordinates the movement of BH, which locates near the active site and separates a single-stranded tDNA at the active site from a downstream double-stranded DNA [28–33] (Figure 1B).

Figure 1. Structural overview of T. thermophilus RNAP-σ^A holoenzyme.

A) Orthogonal views of T. thermophilus RNAP-σ^A holoenzyme – DNA complex. RNAP subunits and DNA are shown as a surface model and labeled (α,α’: light gray, β: cyan, β’: pink, ꞷ: dark gray, σ^A: orange, template DNA: dark green, non-template DNA: light green). Locations of the DNA binding main channel, the NTP entry secondary channel, and the product RNA exit channel are indicated.

B) Close up view of the RNAP active site. β subunit is removed for clarity. The bridge helix (BH, red), trigger loop (TL, blue), DNA and active site Mg (yellow sphere) are shown and indicated.

Biochemical methods provide invaluable insights in deducing mechanisms of transcription. For example, kinetics of transcription and dynamics of RNAP motion have been investigated by quantification of ³²P -labeled RNA separated by gel electrophoresis and fluorescence resonance energy transfer (FRET) using fluorescence labeled RNAP and/or DNA, respectively [34–39]. However, these assays require labeling for detections and intensive control experiments to validate experimental results. The analysis is also often hampered by heterogeneities of RNA product and RNAP conformation as well as unsynchronized reaction. Determining atomic-resolution structures of RNAP by X-ray crystallography and cryo-electron microscopy (Figure 2) provide unprecedented detail of transcription reaction and provide valuable platforms to explain the results of biochemical experiments.

Figure 2. Representative electron density maps of the RNAP active site engaged in the NTP binding and RNA synthesis captured by time-dependent soak-trigger-freeze X-ray crystallography [51].

A) The 2Fo-Fc electron density map corresponding trigger loop of RNAP, DNA, RNA, incoming NTP and Mg²⁺ are shown as gray mesh (1.0 σ) together with the final model of tDNA (green) and TL (blue) (PDB: 6OY5).

B) The 2Fo-Fc electron density map showing PPi remains binding at the active site of RNAP after RNA synthesis (PDB: 6OY7).

RNAP from thermophilic bacteria including Thermus aquaticus and Thermus thermophilus (Figure 1) has been a primary choice for crystallization due to its thermostability [4, 40–44]. Later, Escherichia coli RNAP became an alternative for crystallization, which can investigate not only wild-type but also RNAP derivatives prepared by a plasmid-based over-expression system [45–48]. A crystalline state of bacterial RNAP and DNA complex contacts with neighboring molecules, it remains active for RNA synthesis and undergoes conformational changes during the nucleotidyl transfer reaction. Solvent contents of bacterial RNAP and DNA complex crystals are in range of 50 – 80%, which can be used as a channel for delivering rNTP substrates to the RNAP active site (Figure 3A). The substrate delivery is achieved by simply soaking the RNAP-DNA complex crystal into a cryoprotectant solution containing desired substrates and co-factors (e.g., Mg²⁺), and RNAP synthesizes RNA in crystallo.

Figure 3. Experimental scheme of the time-dependent soak-trigger-freeze X-ray crystallography.

A) A fully grown T. thermophilus RNAP – DNA complex crystal showing the arrangement of molecules (green) inside of the crystal with solvent space (white) between molecules for delivering substrates.

B) A scheme of time-dependent soak-trigger-freeze X-ray crystallography experiment. Multiple RNAP-DNA complex crystals are grown in a hanging drop. The crystals are transferred into a cryoprotectant such as butanediol or glycerol. The crystals are sequentially transferred into the same cryoprotectant containing rNTPs to trigger in crystallo transcription and frozen by liquid nitrogen (LN₂) at desired time points.

To observe real-time motions of enzymes during their reactions, structural biologists including us have combined X-ray crystallography and rapid mixing kinetic techniques to employ an experimental approach called “Time-dependent soak-trigger-freeze X-ray crystallography” also known as in crystallo transcription reaction (Figure 3B). Due to the constraints formed by interactions between neighboring molecules, the enzyme-substate interaction obeys classical “lock and key model” instead of “induced fit” catalytic reaction thereby the reaction speed of in crystallo transcription becomes substantially slower (seconds to minutes) than in solution (milliseconds). Furthermore, highly concentrated RNAP and DNA complex (5 mM) in the small volume of crystals (~0.5 nl) allows complete diffusion of substate to all RNAPs in the crystal before the reaction starts [8]. Therefore, in crystallo transcription reaction provides opportunities of trapping elusive intermediates and determining the atomic resolution structures that are not easily observed by other methods.

Experimental procedure of the time-dependent soak-trigger-freeze X-ray crystallography is simple and doesn’t require any special equipment: crystals of RNAP-DNA complex are sequentially transferred into a cryoprotectant solution followed by exposing the crystals into a same cryoprotectant solution plus rNTPs that delivers rNTPs to the RNAP active site and triggers RNA synthesis (Figure 3B). The reaction is stopped by freezing the crystals with liquid nitrogen at desired time points (seconds to minutes). After X-ray data collection and quick structure determination of the RNAP-DNA complex by molecular replacement, new electron densities corresponding to NTP and RNA appeared at the RNAP active site at the different time points allow us to determine the substrate binding and/or RNA extension. Further structure refinement of the RNAP-DNA complex can capture the conformational change of RNAP and the DNA translocation during the substate binding and RNA synthesis (Figure 2).

Using the time-dependent soak-trigger-freeze X-ray crystallography with the T. thermophilus RNAP and DNA complex crystal, we have imaged many key moments of the RNA synthesis for understanding the molecular mechanism of nucleotidyl transfer reaction such as NTP and catalytic metal bindings at the active site of RNAP, RNA extension and PPi formation as well as the conformational change of RNAP and DNA [8]. We also determined a structure of T. thermophilus RNAP-DNA complex engaged in reiterative transcription [49] which showed an unique RNA extension pathway for the first time to understand how RNAP carries out unusual RNA extension reaction [50]. In a following study, early stages of reiterative transcription complexes were solved by time-dependent soak-trigger-freeze X-ray crystallography which showed an alternative base-paring that allows unusual reiterative transcription [51]. These two studies expanded our understanding of the flexibility of transcription and shed light on the mechanism of transcript slippage.

Radioactively or fluorescence labeled nucleotides have been used in biochemical experiments for detecting RNA synthesis. In contrast, time-dependent soak-trigger-freeze X-ray crystallography can use any nucleotides of interest since the substate binding and RNA synthesis can be visualized as electron density of atomic-resolution X-ray structure. For example, a recent comparative analysis of RNAP-DNA complex with CMPCPP, 2’-dCTP and 3’-dCTP binds at the active site of RNAP revealed that a conserved Arg residue (β ‘Arg425) interacts with a different position of OH group of a sugar moiety of nucleotide and thus favor a different sugar conformation [52]. This work demonstrated how multi-subunit cellular RNAPs select a cognate ribonucleotide during the nucleotidyl transfer reaction.

A simple X-ray crystallographic approach described here is able to trace any enzyme reaction pathway at ambient temperatures without any modification of enzyme and substrate. This method has been used for other polymerases including virus and became a general method for directly looking into biological reactions [8, 23, 53–56].