Volume 10, Issue 5, Pages 1151-1162 (November 2002) Shortening of RNA:DNA Hybrid in the Elongation Complex of RNA Polymerase Is a Prerequisite for Transcription Termination  Natalia Komissarova, Jodi Becker, Stephanie Solter, Maria Kireeva, Mikhail Kashlev  Molecular Cell  Volume 10, Issue 5, Pages 1151-1162 (November 2002) DOI: 10.1016/S1097-2765(02)00738-4

Figure 1 Interactions of RNAP with RNA and DNA in the Processive EC and during Transcription Termination (A) Schematic representation of the EC structure. See details in the text. (B) tR2 terminator of E. coli bacteriophage λ used as a model in our study. The RNA is shown in red; the DNA strands are represented by gray lines. (C) Two forms of the EC at the terminator, one with and the other without the RNA hairpin, were constructed to define the role of the hairpin in the EC destabilization. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 2 The Hairpin Sends a Destabilizing Signal to the RNA:DNA Hybrid in EC (A) The ECs at the wild-type tR2 terminator dissociate within 20 s. In Figures 2A, 2B, and 3B, the top strands of indicated promoter templates downstream from start point of transcription are shown in black. The line above the sequences of the templates indicates the hairpin-encoding region. The symbols in the brackets above the sequences ([h−5] etc.) indicate the template positions relative to the end of the hairpin-encoding sequence. The numbers on the right of the autoradiogram indicate the length of the RNA in nt. Immobilized EC[h−5] containing labeled RNA was chased with 100 μM of all 4 NTPs in transcription buffer (TB) containing 300 mM KCl. After indicated time periods, either the chase was stopped (lanes 2, 3, and 7) or the reaction was fractionated by centrifugation into a pellet and supernatant. Half of the supernatant (“s”) was withdrawn; the rest of the material (“p”) contained the remaining half of the supernatant and pellet. The fractions were analyzed on a denaturing polyacrylamide gel. In a completely dissociated EC, the RNA is equally distributed between “s” and “p” fractions. In lane 6, EC [h−5] was treated with RNase T1 (symbolized by scissors) to truncate the hairpin before the chase (lane 7). (B) EC8UUUUUAUU dissociates in 30 min. The substitutions made to the tR2 hairpin are underlined in the sequence of template tR2mut. Lanes 1 and 2: chase of EC[h−10] on template tR2mut (100 μM NTPs, 300 mM KCl) followed by the “s”/“p” fractionation shows that within 20 s the ECs dissociate completely from the mutant tR2 terminator. Lanes 3–12 and the scheme: obtaining EC8UUUUUAUU. EC[h+20] was labeled in oligoU tract (*), treated with RNase T1, washed, and treated with pyrophosphate (PPi). After pyrophosphate was washed off, indicated NTP subsets (5 μM) were added for 5 min to map the RNA 3′ end. Lanes 13–20: The 8, 9, and 12 nt RNA species were eluted from PAAG of Figure 2B (lane 5) and treated with RNase T1 (500 U/μl, 1 min) or RNase A (1 μg/μl, 5 min). Lanes 21–32: the complexes of lane 5 were destabilized by elevating KCl concentration to 300 mM. The “s” and “p” fractions were collected at the indicated time points. 400 μM ATP was present in this stability test to equalize the conditions with the termination assay, where each of the 4 NTPs was present in 100 μM concentration (Figures 2A and 2B, lanes 1 and 2). Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 3 RNA Sequence Downstream from the Hairpin Affects Stability of the EC (A) Decrease in rU:dA content of the RNA:DNA hybrid inhibits dissociation of the EC. The ECs with 8 nt RNA were obtained as described in supplemental data (http://www.molecule.org/cgi/content/full/10/5/1151/DC1) and destabilized by 300 mM KCl, “s” and “p” fractions were collected at the indicated time points. The fraction of dissociated RNA was calculated as [2 × s/(s+p)] × 100%. (B) Completely formed hairpin destabilizes ECs halted 7 and 8 nt downstream even in the absence of the oligoU tract. The indicated complexes containing full-size or truncated hairpin were obtained on the tR2U-less template by walking, challenged by KCl, and fractionated. (C) The strengthening of the upstream part of the RNA:DNA hybrid, but not of the middle part of the hybrid, countervails the destabilizing action of the termination hairpin. The A to C substitutions in the RNA, made to reinforce the hybrid in EC[h+8], are underlined. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 4 RNA Hairpin Destabilizes the EC by Melting the RNA:DNA Hybrid (A) Assembled ECs terminate properly at the tR2 signal. Immobilized EC[h−5] was obtained with RNA oligo 10 and DNA oligos tR2 as described in Experimental Procedures. EC[h−5] was chased and fractionated as in Figure 2A. Here and in Figure 4B, the complete sequences of DNA and RNA oligos used for assembly are shown. Note that template and nontemplate DNA oligos were completely complementary to each other; however, the hybrid between RNA oligo 10 and template DNA oligo had two mismatches (CC sequence at 3 and 4 nt from the 5′ end of the RNA). The mismatches did not affect the complex formation. They were introduced to disrupt the strong hairpin in the DNA oligos and prevent it from interfering with the EC assembly. (B) Obtaining the assembled ECs for stability test. ECs were assembled with DNA oligos tR2U-less in TB containing 40 mM KCl. EC4-8 were obtained with RNA oligos 3 to 7 and labeled with [α-32P]-ATP at the RNA 3′ end. The autoradiogram shows an example of a walking experiment: EC5 was incubated with 5 μM of the indicated NTP subsets for 5 min. To obtain EC[h+8], the initial EC was assembled with RNA oligo 10 and walked to position [h+8]. (C) The shortening of RNA:DNA hybrid in the EC to 5 bp mimics the action of the hairpin. The ECs described in (B) were treated with 300 mM KCl and fractionated at the indicated time points. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 5 Potassium Permanganate Footprinting of the Template DNA Strand in the Vicinity of the tR2 Hairpin ECs labeled in both the RNA and the DNA were obtained using the tR2U-less template and treated with 10 mM KMnO4 for 30 s (see supplemental data at http://www.molecule.org/cgi/content/full/10/5/1151/DC1). In lane 6, the hairpin was truncated as described in Figure 2A and the EC was walked to [h+8] position. All T residues are shown in green. The asterisks indicate 5′ labeling of the template DNA strand. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 6 Pol II ECs Terminate at the tR2 Signal and Are Destabilized by the Hairpin (A) The initial EC was assembled with DNA oligos tR2 and walked to the indicated positions. Where marked, the complexes were treated with RNase T1 and washed. The ECs were chased and fractionated as in Figure 2A. (B) The initial EC was assembled with DNA oligos tR2U-less, walked to [h+2] position, was or was not treated with RNase T1, was walked further, and halted at the indicated positions downstream from the hairpin. ECAAUAGCCA was assembled with RNA oligo 7. The ECs were challenged with 300 mM KCl, and dissociation was quantified. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)

Figure 7 Hairpin Action during Termination (A) Coupling between the hairpin formation and the melting of the RNA:DNA hybrid in the EC. In this panel, as opposed to (B) and all other Figures, transcription proceeds from right to left, to display melting of the RNA:DNA hybrid more clearly. The RNA and transcribed DNA strand are shown in red and black, respectively. The gray irregular shape symbolizes the RNAP domain(s) proposed to interfere with the folding of the hairpin. Red arrows indicate direction of the movement of the RNA hairpin upon completion of the stem. The motions of the hairpin are also shown schematically. The model is based on a combination of two X-ray structures: of the RNA hairpin with a tetraloop (Wu et al., 2001) and of the RNA:DNA hybrid, taken from the structure of the Pol II EC from S. cerevisiae (Gnatt et al., 2001). (B) The consecutive steps in intrinsic transcription termination in E. coli. See the explanation in the Discussion. Molecular Cell 2002 10, 1151-1162DOI: (10.1016/S1097-2765(02)00738-4)