1. Modification of the Properties of Elongating RNA Polymerase by Persistent Association with Nascent Antiterminator RNA 
Ranjan Sen, Rodney A King, Robert A Weisberg 
Molecular Cell 
Volume 7, Issue 5, Pages (May 2001)
DOI: /S (01)00243-X

2. Figure 1
Stalling and Restarting Elongation after Transcription of putL
(A) A PCR fragment (bold horizontal line) containing the λ TR′ terminator and lac operator downstream of the HK022 PL promoter and putL site served as a template to monitor the effects of oligonucleotides complementary to putL RNA on terminator readthrough. Translocation of the elongation complex (open oval) was blocked by the Lac repressor (shaded oval) bound to the lac operator. The two stem-loops indicate the nascent putL transcript bound to RNAP (see text). The promoter-proximal end of the template was biotinylated so that the DNA could be immobilized by binding to a streptavidin coated magnetic bead. Distances and transcript sizes are indicated in nucleotides below the cartoon. A similar template without a terminator and with a shorter putL-lac operator distance (89 nt) was used for RNaseH and footprinting assays (see Methods).
(B) The template lacking the terminator was transcribed in the presence of [α-32P]CTP and Lac repressor. After 2 min, an aliquot was removed to assess roadblocked complex, and the remainder was chased by adding IPTG and incubating an additional 3 min. The transcripts made in the absence and presence of IPTG (−IPTG and +IPTG lanes, respectively) were fractionated according to size (size increases from left to right) and scanned. RB, road-blocked transcript; RO, run-off transcript
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

3. Figure 2 The putL Site Is Dispensable after It Is Transcribed
Stalled elongation complexes were formed on the template that contained TR′ (Figure 1A) in the presence of unlabeled NTPs. One aliquot of the reaction (“uncleaved”) was chased with IPTG, NTPs, and [α-32P]CTP. A second aliquot was cleaved at a Bbv1 site (Figure 1A) for 5 min at 37°C. Magnetic beads were used to separate the cleaved aliquot into bound and supernatant fractions, and these fractions were chased as indicated above. The extent of DNA cleavage was monitored in a parallel reaction in which the stalled elongation complexes were not chased. The labeled RNA and unlabeled DNA were fractionated according to size in appropriate gels and visualized by Ethidium bromide staining (DNA gel) or phosphorimaging (RNA gel).
(A) DNA gel. Lane M: DNA markers, sizes in bp; Lane 1: Uncleaved aliquot; Lane 2: Cleaved aliquot, supernatant + bound fractions; Lane 3: Cleaved supernatant fraction.
(B) RNA gel. Lane 1: Uncleaved aliquot; Lane 2: Cleaved supernatant fraction; Lane 3: Cleaved bead-bound fraction. Bbv1 cleavage products (DNA gel) are indicated by the arrow. Percent readthrough (%RT) is shown below the RNA gel
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

4. Figure 3 Footprinting putL RNA in Stalled Elongation Complexes
(A) RNase V1 cleavage pattern. The stalled elongation complexes were formed by transcribing the lac operator template that lacks a downstream terminator with wild-type RNAP (left) or RNAP β′-Y75N (right) in the presence of Lac repressor. The end-labeled transcripts were fractionated by size on a sequencing gel and scanned. Uncut lane: No treatment before gel analysis. RB lane: The complex was treated with RNase V1 before gel analysis. Free lane: The RNA was extracted from the complex and then treated with RNase V1 before gel analysis. M lane: Roadblocked RNA was partially hydrolyzed with alkali before gel analysis. The colored tracings to the right of each scanned image show the intensities of the bands in the RB (blue), Free (red), and Uncut (black) lanes. The drawings next to the tracings indicate the positions of stem-loops 1 and 2. Concentrations of RNAP, DNA, and Lac repressor were 20 nM, 75 nM, and 40 nM, respectively.
(B) RNase T1 cleavage pattern. The conditions are those described in panel (A), except that RNase T1 was used
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

5. Figure 4
Summary of the RNase V1 and T1 Protection Patterns of putL RNA in the Stalled Elongation Complexes Made with Wild-Type (Top) or β′-Y75N (Bottom) RNAPs
The protected areas of putL RNA are shown by gray shading superimposed on the predicted secondary structure of the RNA. The intensity of the shading indicates the strength of protection. Mutations of unpaired residues that reduced antitermination are indicated by red circles, and those of paired residues by red rectangles (King et al., 1996; Banik-Maiti et al., 1997). The latter mutations were compensated by changes in the opposite strand that restored pairing. The red and blue arrows indicate bases hypersensitive to RNases V1 and T1 cleavage, respectively, in the roadblocked complexes
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

6. Figure 5
Inhibition of put RNA Function by Hybridization with Complementary Oligonucleotides
(A) The oligonucleotides are represented by dashed lines with arrowheads at the 3′-ends and are aligned to complementary regions of the putL transcript. The putL RNA secondary structure is drawn according to (Banik-Maiti et al., 1997).
(B) The indicated oligonucleotide was added to stalled elongation complexes 2 min after transcription initiation and allowed to hybridize for 5 min at 37°C. The complexes were then chased by adding IPTG, NTPs, and [α-32P]CTP. The concentration of each oligonucleotide was 3 μM, except for oligonucleotide IA, which was 1 μM. The concentrations of DNA and RNAP were 12 nM and 50 nM, respectively. The percent readthrough (%RT) is shown below each lane
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

7. Figure 6
RNase H Cleavage of Roadblocked Complexes Treated with Different Oligonucleotides
The roadblocked complexes were formed by transcribing the lac operator template that lacked a downstream terminator in the presence of Lac repressor and [α-32P]CTP. After allowing oligonucleotide hybridization, the resulting RNA-DNA hybrids were cleaved with RNase H. (A) Roadblocked complexes formed with WT polymerase; (B) roadblocked complexes formed with RNAP β′-Y75N; (C) RNA was extracted from the roadblocked complexes formed with WT polymerase before the addition of oligonucleotides and RNase H; and (D) roadblocked complexes were formed on a template containing the stem-loop 1 flip mutation (see text), but the conditions were otherwise those of (A). The concentrations of oligonucleotides, DNA, and RNAP were as described in the Figure 5B legend. RNase H concentration was ∼0.12 units/μl. The position of the RNA in the roadblocked complex is indicated as RB and the shortened transcripts are primarily the cleavage products of the RB RNA
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

8. Figure 7
Inhibition of Antitermination Function by Complementary Oligonucleotides When Added at the Time of Transcript Initiation
The template shown in Figure 1A was transcribed for 15 min in the presence of [α-32P]CTP and heparin but without Lac repressor. Concentrations of DNA and RNAP were the same as in Figure 5. Percent readthrough (%RT) of the TR′ terminator is indicated below each lane
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)

9. Figure 8 Cleaving the Tether between Nascent putL RNA and the 3′-End
(A) The cartoon shows the design of the experiment (see legend to Figure 1A for additional details). The upstream end of the template (shaded circle) is bound to magnetic beads to facilitate the separation of released and retained RNA after cleavage of the tether at the position of oligonucleotide C by RNase H. The concentrations of DNA, RNAP, and Lac repressor were 15 nM, 50 nM, and 40 nM, respectively.
(B) The template of (A) was transcribed in the presence of [α-32P]CTP and Lac repressor. An aliquot was treated with oligonucleotide C and RNase H, and separated into supernatant and pellet fractions. The RNA was fractionated in a denaturing gel and scanned. Input: Roadblocked transcript (RB) before RNase H cleavage. Sup: Roadblocked transcript after RNase H cleavage and release from magnetic beads. Pellet: Roadblocked transcript cleaved by RNase H and retained on magnetic beads after three washes with T buffer (200 μl each wash). The 3′ cleavage product is too small to be seen on this gel.
(C) Unlabeled, stalled elongation complexes were chased with IPTG in the presence of [α-32P]CTP and fractionated in a denaturing gel. The presence or absence of oligonucleotide C, RNase H, and washes of the magnetic beads with T buffer are indicated above the scanned image, and the percent readthrough (%RT) of the TR′ terminator is indicated below. The positions of the terminated (TR′) and runoff (RO) bands for uncleaved and cleaved transcripts are indicated to the left and right of the scanned image
Molecular Cell 2001 7, DOI: ( /S (01)00243-X)