Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase

Abstract

Time-resolved characterization of T7 RNA polymerase pausing and terminating at a class II termination site has been carried out using site-specifically tethered chemical nucleases. The data indicate that T7RNAP normally moves uniformly down the template as a rigid body. However, at the class II site this movement is interrupted, and the leading edge of the polymerase moves further along the DNA than the trailing edge. This discontinuous movement may persist until it can no longer be accommodated by conformational changes in the elongation complex, at which point the polymerase can either pause or terminate. Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase fingers and thumb subdomains. The introduced cysteines disrupt a thumb-fingers salt-bridge and, under reducing conditions, this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. These observations suggest that termination requires that the thumb and fingers subdomains move apart, in a reversal of a conformational change important for initially forming a stable transcription complex.

Fig. 1. (A) Left: transcript sequence of the 66 bp template used in these studies (the sequence from –1 to –17 is that of a consensus T7 promoter). The invariant class II element is highlighted in magenta. Right: gel analysis of transcription on this template. Lane 1: GTP, ATP and [α-³²P]GTP only added; transcription halts at +14, misincorporation allows some transcription to +15. Lanes 2–5: UTP+CTP chase of reaction from lane 1 for 10 s to 20 min, as indicated. R.O.: runoff. (B) Kinetics of Fe-BABE transcript cleavage. Halted ECs containing a 16 nt transcript labeled with [α-³²P]CMP at its 3′-end were formed with T7RNAP conjugated with Fe-BABE at residue 388 (lane 1). Transcript cleavage was induced by addition of H₂O₂ and ascorbate to generate hydroxyl radicals either after (lane 2), simultaneous with (lane 3), or varying times before (5 s to 16 min as indicated), addition of quenching buffer.

Fig. 2. (A) Non-template (lanes 1–9) or template (lanes 10–13) strand cleavage by Fe-BABE conjugated to aa 388 in ECs halted at +15 (lanes 1 and 10), or +19 (lanes 2 and 11), or chased from +14 with UTP+CTP for the indicated times. Cleavage sites were mapped by reference to G+A ladders prepared with the identical DNAs and are highlighted by vertical lines. (B) Structure of the T7RNAP EC (Yin and Steitz, 2002; pdb:1msw) with the template strand in cyan, the non-template strand in blue, and the RNA in red. Residues 388 and 385 are in magenta and regions of the template and non-template strands cut by the 388 conjugate (in the +15 and +19 halted complexes) are colored yellow and green, respectively. Note that the DNA labeled as ‘Modeled’ was added onto the crystal structure by extending the upstream DNA. (C) As in (A), but for complexes with the conjugate at residue 385.

Fig. 3. As in Figure 2A, but for conjugates at residues 153 (A), 239 (B), 602 (C), 711 (D), 713 (E) and 745 (F). Conjugate positions are labeled and highlighted in magenta on the polymerase structures, and cleavage positions (for the +15 and +19 complexes) are in yellow and green for the template and non-template strand, respectively. Note that, with the exception of the structure in (C), DNA extensions have been modeled onto the EC structure to allow display of cleavage positions lying up- or downstream of the visible DNA in the EC crystal structure.

Fig. 4. (A) Denaturing PAGE of the 5′-end labeled template used in these studies. Lane 1: DNase I-treated DNA alone. Lane 2: DNase I-treated DNA with T7RNAP EC halted at +19. Lane 3: DNase I-treated DNA with T7RNAP halted at +15. Lane 4: DNase I-treated DNA with T7RNAP paused at the class II site. Lane 5: DNA alone (no DNase I). (B) Superimposed scans of lanes 1 (black) and 3 (red) from (A). Horizontal line indicates a region of >80% protection. (C) Superimposed scans of lanes 1 (black) and 2 (green) from (A). Horizontal line indicates a region of >80% protection. (D) Superimposed scans of lanes 1 (black) and 4 (blue) from (A). Horizontal line indicates region of >50% protection. Arrows in B–D indicate positions of enhanced DNase I sensitivity.

Fig. 5. (A) Halted (lanes 1 and 2) or paused (lanes 3 and 4) complexes containing transcripts internally labeled at +19 were formed on a template containing a non-functional class II element (lanes 1 and 2) or a functional class II template (lanes 3 and 4). Halting on the non-functional class II template was effected by incorporation of 3′-dCMP at +31. Peroxide and ascorbate were added to induce cleavage by the conjugate at residue 388 (lanes 2 and 4, labeled ‘+’). The transcript in the halted complex is cleaved 5 nt away from the 3′-end (lane 2), while in the paused complex it is cleaved 8 nt away. (B) Crystal structure of the T7RNAP EC with NT (non-template) strand, T (template) strand and residues 385, 388 colored as in Figure 2B. The –5 nt of the transcript is highlighted in yellow.

Fig. 6. (A) Structure of the T7RNAP EC (pdb:1msw) with the thumb and fingers subdomains in cyan and orange, respectively. Residues D660 and R379, which form a salt-bridge between the fingers and thumb, are labeled and highlighted in red and blue respectively. (B) Denaturing PAGE of transcription reactions run by forming complexes halted at +14 and then chased with CTP and UTP for 10 s (odd numbered lanes) or 10 min (even numbered lanes) with either wild-type protein (lanes 1 and 2), D660C/R379C double mutant under reducing conditions where the 379–660 disulfide bond is absent (lanes 3 and 4), or the double mutant under conditions which induce formation of the disulfide bond (lanes 5 and 6).

Fig. 7. Complexes halted at +14 were chased for the indicated times either with 1 mM CTP and 0.5 mM UTP (lanes 1–6),10 µM CTP and 0.5 mM UTP (lanes 7–12), 10 µM CTP and UTP (lanes 13–18) or 1 mM CTP and 10 µM UTP (lanes 19–24). R.O., runoff.

Fig. 8. A model for class II termination. 1. EC crystal structure (pdb: 1HW38; duplex DNA extensions are modeled onto the DNA from the crystal structure and the RNA is omitted for clarity). Polymerase movement is in the direction of the lower arrow. The thumb (aa 330–410, red), fingers (aa 560–680, blue) and N-terminal (aa 60–255, green) domains form a box around the DNA which, barring a conformational change, effectively precludes template release. If the EC is at +25 on the template used in these studies, then the region highlighted in magenta corresponds to the conserved class II element at +16 to +23. 2. Hypothetical +30 Paused EC structure. Interactions between 385/388 and the class II element, first established in the +25 EC, may persist as the RNA is extended to +30. The additional DNA is accommodated in the EC by a combination of ‘scrunching’ and/or conformational changes in the polymerase, and, after reaching +30, the polymerase backtracks ∼3 nt to form the stably paused complex. The N-terminal domain may shift and rotate as suggested by the blue arrow, so that an Fe-BABE conjugate at 239 would cleave upstream of the class II element. This movement of the N-terminal domain could also create room to allow the trailing edge of the transcription bubble to become duplex, as has been observed during pausing at the class II site (Song and Kang, 2001). 3. Spontaneous release of the class II element leads to escape from the pause and recovery of the normal EC structure (now shown at +31). 4. Strain in the paused complex may cause the thumb and fingers to move apart as suggested by the white arrows, so the RNAP can release DNA and terminate transcription.