1. The Mechanism of Intrinsic Transcription Termination
Ivan Gusarov, Evgeny Nudler 
Molecular Cell 
Volume 3, Issue 4, Pages (April 1999)
DOI: /S (00)

2. Figure 1 Characterization of the Trapped Termination Complex
(A) RNA sequence of the tR2 terminator with termination sites indicated by arrowheads.
(B) Trapping TEC at the terminator. TEC20 carrying [32P]RNA and [32P]DNA was immobilized on Ni-NTA-agarose beads and then chased through the terminator in 150 mM KCl TB (lanes 1 and 2) or 5 mM KCl TB (lanes 3–7, 9, and 10). After 10 min of chase, reaction beads were washed with 150 mM KCl TB (lane 2) or 5 mM KCl buffer (lanes 4 and 5). The effect of a high dose of GreB on TECU7 (TEC68) is shown in lane 5. After GreB treatment, the truncated complex (TEC56) was washed with 150 mM KCl TB. Lanes 6–8 demonstrate the RNA and the DNA recovered from TECU7, which was first obtained in 5 mM KCl solution followed by loading on beads. Prepared this way, TECU7 was washed with the 5 mM KCl TB (lane 6) or 150 mM KCl TB (lane 8) or treated with GreB (lane 7). %T represents the termination efficiency.
(C) Role of the hairpin in trapping TEC. Schematic representation of the tR2 hairpin destabilized by inosins (boldface I) incorporated into specific positions of the stem during walking reactions (top) or by the 14 nt DNA oligonucleotide (oligo #1) annealed to the left half of the stem (bottom). The inhibitory effect of inosines or oligo #1 on trapped TECU7 formation is demonstrated by lanes 9 and 10 of (B).
Molecular Cell 1999 3, DOI: ( /S (00) )

3. Figure 2
Effect of the Termination Hairpin on Protein–RNA Interactions in TEC
(A) RNA sequences of modified tR2 terminators. Boxed nucleotides denote changes from wild type made to incorporate 4-thio-UMP (boldface U’s) or 6-thio-GMP (boldface Gs) at indicated positions during walking reaction. Boldface C stands for the substitution that allows the stalling of active TEC precisely at position U7.
(B) The top panel (12% urea-PAGE) shows [32P]RNA transcripts from derivatized TECU7 before or after challenge with a mixture of UTP, GTP, and CTP (UGC, lanes 2, 4, 6, and 8). Oligo #1, which prevented trapping, and control oligo #2 were used as indicated. Negative numbers denote the distance of cross-linkable 4-thio-U (lanes 1–4) or 6-thio-G (lanes 5–8) from the RNA 3′ end. The bottom panel (4% SDS-PAGE) shows protein-[32P]RNA cross-linking products (bands 1, 2, and 3) from active (lanes 1 and 5) and trapped (lanes 3 and 7) TECU7.
(C) Mapping of RNA cross-links in β′ and β subunits, obtained with the 4-thio-U (−12) probe, by single-hit cleavage at methionine and cysteine residues. Autoradiograms of gradient (7% to 14%) SDS-PAGE show products of partial degradation with CNBr or NTCBA of the cross-linked products from (B). Bar columns present theoretical patterns of the NH2-terminal (N) and COOH-terminal (C) Families of methionine and cysteine fragments, with numbers indicating residue positions in β or β′ polypeptide. As the reference marker, CNBr degradation products of the β subunit labeled near M1304 are shown in the lane “marker.”
(D) Summary of RNA cross-linking mapping results obtained with 4-thio-U (-12) probe (left) and 6-thio-G (−8) (right). Horizontal lines symbolize 1407–amino acid β′ polypeptide (top) or 1342–amino acid β polypeptide (bottom). Locations of cross-linking sites are indicated by arrowheads. The size of arrowheads reflects a relative intensity of the cross-linking.
Molecular Cell 1999 3, DOI: ( /S (00) )

4. Figure 3
Effect of the Termination Hairpin on the RNA–DNA Hybrid in TEC
(A) RNA sequences of modified tR2 terminators. Boxed nucleotides denote changes from wild type made in order to incorporate U· probe at indicated positions during walking reaction. Boldface C stands for the substitution that allows the stalling of active TEC precisely at position U7.
(B) RNA–DNA cross-linking in TECU7. The autoradiogram shows free [32P] transcripts (bottom panel) and cross-linked DNA-[32P]RNA or protein-[32P]RNA products (top panel) recovered from active (lanes 1, 3, and 5) or trapped TECU7 (lanes 2 and 4). Negative numbers show the distance of U· from the RNA 3′ end. Oligo #1, which prevented trapping of TECU7, and control oligo #2 were used as indicated. The bottom part of the gel was underexposed to compensate for low yield of the cross-linking species.
(C) Potassium permanganate footprinting of the nontemplate DNA strand in TECU7 and nearby complexes. Modified single-stranded thymidines involved in the transcription bubble are indicated.
Molecular Cell 1999 3, DOI: ( /S (00) )

5. Figure 4 Hairpin Formation as a Function of RNA–DNA Hybrid Stability
(A) Oligo/hairpin competition assay. On the left is schematic representation of the competition between oligo #1 and the hairpin. Oligo #1 cannot be annealed to the hairpin RNA if it is added after TEC reaches the U7 termination point (top). However, annealing does occur if the oligo is added to TEC56 followed by walking TEC to position U7 (bottom). Arrows denote RNAse H cleavage sites. The panel on the right shows the described experiment. Decrease of the intensity of 68 nt RNA band and appearance of shorter products (lane 4) are indicative of RNAse H cleavage.
(B) Monitoring the hairpin formation on modified tR2 templates. RNA sequences of modified T stretches of tR2 are shown on the top of the panel. Boxed nucleotides denote changes from wild type. Oligo #1 followed by RNAse H were added after walking [32P]TEC reached the position indicated by the arrowhead and washed with 5 mM KCl TB. Substantial decrease of the intensity of the 68 nt band (lanes 2 and 8) or the 66 nt band (lane 20) is indicative of RNAse H cleavage. UTP and CTP (30 μM) were added to TECU7 as indicated for 5 min. UTP, CTP, and ATP were added to TECU5 (lane 21).
(C) Monitoring hairpin formation by protein–RNA cross-linking. The bottom panel (12% urea-PAGE) shows [32P]RNA transcripts from derivatized TECU7 obtained on control (lane 2) and modified tR2 templates (lane 1). Negative numbers indicate the distance of cross-linkable 4-thio-U from the RNA 3′ end. The top panel (4% SDS-PAGE) shows β′-[32P]RNA (bands 1 and 2) or α-[32P]RNA cross-linking products (band 3).
Molecular Cell 1999 3, DOI: ( /S (00) )

6. Figure 5 Role of the T Stretch in Pausing at the Terminator
(A) Effect of point substitutions in the distal portion of the tR2 T stretch on termination. Preformed [32P] TEC20 was chased for 30 s with 1 mM NTP through the wild-type (lane 1) or mutant tR2 terminators (lanes 2–6). The bottom table represents termination efficiencies (%T) at individual template positions.
(B) Pausing (P) at the wild-type T stretch (lanes 7–10) and the T stretch with the C8 substitution (lanes 11–14) during elongation with 1 mM NTP. Samples were taken at the time (in seconds) indicated above each lane.
(C) Summary graph. For each modified template, the half-life of the pause and the efficiency of termination at position 7 were determined and plotted as a fraction of those for wild-type T stretch template. The error bars show the standard deviation for three independent experiments for each template.
Molecular Cell 1999 3, DOI: ( /S (00) )

7. Figure 6 The Mechanism of Termination
(A) Stable TEC paused at the end of the U stretch of tR2 terminator. Lines indicate protein regions, mapped by cross-linking (Nudler et al. 1998), that are involved in formation of the RNA–DNA hybrid–binding site (HBS), the RNA-binding site (RBS), and the duplex DNA-binding site (DBS). β′ is green, and β is blue.
(B) Conversion of the paused TEC into the unstable trapped configuration. Formation of the hairpin leads to: TEC inactivation (illustrated by the cross over the catalytic site), disruption of four to five base pairs of A:U hybrid in HBS, closing of the DNA bubble from behind, displacement of RNA from RBS, and, presumably, conformational change in DBS triggering DNA release.
Molecular Cell 1999 3, DOI: ( /S (00) )