Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation

Abstract

Regulatory sequences or erroneous incorporations during DNA transcription cause RNA polymerase backtracking and inactivation in all kingdoms of life. Reactivation requires RNA transcript cleavage. Essential transcription factors (GreA and GreB, or TFIIS) accelerate this reaction. We report four cryo-EM reconstructions of Escherichia coli RNA polymerase representing the entire reaction pathway: (1) a backtracked complex; a backtracked complex with GreB (2) before and (3) after RNA cleavage; and (4) a reactivated, substrate-bound complex with GreB before RNA extension. Compared with eukaryotes, the backtracked RNA adopts a different conformation. RNA polymerase conformational changes cause distinct GreB states: a fully engaged GreB before cleavage; a disengaged GreB after cleavage; and a dislodged, loosely bound GreB removed from the active site to allow RNA extension. These reconstructions provide insight into the catalytic mechanism and dynamics of RNA cleavage and extension and suggest how GreB targets backtracked complexes without interfering with canonical transcription.

Keywords: GreB; RNA polymerase structure; backtracking; cryo-EM; transcription; transcriptional arrest; transcriptional rescue.

Figure 1. Schematic of transcription, RNAP, and GreB.

(A) Nucleotide addition and backtracking: In the post-translocated state (POST), the RNA 3′ end occupies the P-site, while the A-site holds the +1 template DNA, which dictates the next rNTP substrate to bind (green, top). Catalysis extends the RNA giving rise to the pre-translocated state (PRE). RNAP translocates relative to DNA by one base pair and concludes the cycle. Misincorporations, weak RNA-DNA hybrids or pause signals can cause backtracking and extrusion of the RNA from the active site (bottom, right). Reactivation requires forward translocation (slow, upward arrow), intrinsic RNA cleavage (slow, diagonal arrow), or Gre-factor assisted cleavage (bottom, left). (B) RNAP structure: View into the secondary channel with the five subunits, DNA, and RNA indicated (top). RNAP modules, which will be referred to throughout the manuscript are indicated (bottom) (C) GreB contains two domains. The N-terminal coiled-coil inserts its tip with two conserved acidic residues into the active site through the secondary channel. The C-terminal domain interacts with the RNAP surface. The electrostatic surface potential of GreB shows one side is highly positively charged and predicted to interact with backtracked RNA (right).

Figure 2. RNAP and active site conformation of backtracked complex.

(A) The swivel module (clamp and shelf domain of RNAP, shades of blue and orange) can rotate relative to the core module by almost 4˚ in the backtracked complex. The trigger loop insertion domain (SI3, orange and blue) and downstream DNA move along with the swivel module. Superposition of EM maps from the two 3D classes (blue 37%, orange 63%) confirms swiveling. (B) Downstream DNA movement affects the downstream end of the RNA-DNA hybrid. The template DNA shifts around 2Å between the two conformations and affects the RNA in A- (+1) and P-site (-1). The two positions are reminiscent of an oscillation around the position observed in an EC (Kang et al., 2017). (C) Cryo-EM density (grey and green transparent surface) for the active site reveals the backtracked RNA at lower contour level (black sticks and green surface), the BH, unfolded TL, and Aspartate triad (pink), the template DNA (orange) and MgI (green). (D) Superposition of a substrate bound EC shows the steric clash between the folded trigger loop (grey) with the backtracked RNA (black) in the present reconstruction.

Figure 3. Active site of pre-cleavage complex and GreB interaction with RNAP.

(A) Cryo-EM density (light grey, transparent surface) for the active site reveals density for the RNA (black) and template DNA (orange), the Aspartate triad and MgI (pink and green respectively) and GreB (cyan). (B) Comparing the backtracked complex (RNA transparent grey) with the pre-cleavage complex (RNA black), shows GreB shifts the backtracked portion of the RNA towards the BH and unfolded TL (pink). (C) A different view of the active site shows well-resolved density (grey transparent surface) for the tip of GreB (cyan). The backtracked RNA base in position +2 (black), stabilizes the backbone of GreB and helps to orient the catalytic residues. (D) GreB (cyan) interacts through the CTD with the secondary channel rim helices (light orange) and the trigger loop insertion SI3 (orange, SI3_pre), which rotates almost 30° relative to the orientation in the backtracked state (blue, SI3_bt). A low-pass filtered map confirms SI3 rotation (orange surface).

Figure 4. Active site of post-cleavage complex and additional GreB binding.

(A) Density for the active site (grey transparent surface) reveals the RNA in a post-translocated register (black), the template DNA (orange) as well as RNAP components (pink). Density for MgI (green) is weak but visible at lower contour level. The tip of GreB (cyan) is more disordered than in the pre-cleavage complex. (B) Similar to the pre-cleavage complex, a low-pass filtered map confirms SI3 rotates relative to the backtracked state as a result of GreB binding. (C) Superposition of pre- (grey transparent) and post-cleavage (colored) complex indicates that GreB disengages from the active site as a result of RNA cleavage. (D) 3D classification indicates a population of RNAP that bound a second copy of GreB (green) close to the upstream DNA, which becomes apparent at low contour level.

Figure 5. Active site of reactivated, substrate bound complex and RNAP conformational changes.

(A) Two thirds of particles, showed density (grey transparent surface) for the substrate and MgII in the active site. The substrate base pairs to the template DNA (orange). The TL (pink) is folded but density for MgI is absent presumably because the RNA (black) lacks a 3′-OH. (B) TL folding repositions SI3, which approaches the secondary channel (compare SI3_EC vs. SI3_react). Likewise, the rim helices approach SI3 and close the secondary channel for GreB access. (C) Movement of the F-loop along with the rim helices establishes new contacts between F-loop and SI3 linkers, which may stabilize the folded TH. (D) Weak density for GreB was apparent on the surface of RNAP, suggesting it can maintain loose interactions with RNAP. Shown here is a 10 Å low-pass filtered map at two contour levels.

Figure 6. Models for RNA cleavage and the role of GreB during transcription elongation.

(A) In the backtracked complex, the nucleophile (red sphere) can be placed in line with the scissile bond. The O4′ of the first backtracked base (+2, black) may help to coordinate the nucleophile. MgII (modelled), could be coordinated by β′-D460, and β′-D462, but would be 3Å away from the attacking nucleophile. Small changes in the RNA backbone conformation may allow to adopt optimal geometry for the nucleophilic attack. (B) In the pre-cleavage complex, the nucleophile (red sphere) can be placed in line with the scissile bond. MgII can be modeled to be coordinated by β′-D460, β′-D462, the phosphate of the base in the A-site (+1) as well as GreB D41 and E44. However, this position (position 1) is 5Å away from the nucleophile. A change in the RNA backbone conformation may allow direct coordination of the nucleophile by MgII. Alternatively, a hypothetical third ion (modelled in position 2) as observed in DNA polymerase could activate the nucleophile. S43 in GreB might help orient the nucleophile consistent with a subtle decrease in cleavage rates upon mutation to Alanine. (C) Model for GreB’s role in transcription elongation: During a canonical elongation cycle (left), GreB cannot access the active site of substrate-bound and pre-translocated complexes because of the folded TH and secondary channel closure (not shown). As a result of erroneous incorporations or pause inducing DNA sequences, RNAP can backtrack and extrude the RNA 3′-end from the active site (right). RNA backtracking by 2 or more bases results in an unfolded TL and allows GreB to access the active site. GreB accelerates RNA cleavage and gives rise to a post-translocated EC, which can resume transcription (bottom).