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Supplemental Results
70 substitution K299E increases utilization of the plac pause element in vivo
To test the effect of 70 substitution K299E on the utilization of the plac
promoter-proximal pause element in vivo, we took advantage of our previous
demonstration (Nickels et al, 2004) that pausing in the promoter-proximal region of plac
can be assayed in vivo by means of a PR'/plac hybrid construct in which the plac initial
transcribed region replaces that of PR'. In the context of the hybrid promoter (fused
together with a downstream terminator to a lacZ reporter gene), Q antiterminator
function depends on the plac pause element. We showed previously that Q stimulated
lacZ expression several fold in cells containing this PR'/plac reporter construct and
lacking a functional greA gene (the removal of GreA in vivo increases the half-life of
promoter-proximal 70-dependent pauses; Marr and Roberts, 2000). We therefore
examined the ability of Q to stimulate lacZ expression in reporter strain cells containing
either chromosomally encoded wild-type 70 or chromosomally encoded 70 K299E (see
Supplemental methods). We found that substitution K299E enhanced the stimulatory
effect of Q (from 3.9-fold to 6.5-fold) (Fig. S1D), consistent with our observation that
the K299E substitution increased 70-dependent early elongation pausing at placUV5 in
vitro. Control assays performed with reporter strain cells bearing a mutated pause
element revealed that the stimulatory effect of Q on lacZ expression was dependent on
the pause element (Fig. S1D).
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70 substitutions A370F and A370Y strengthen the interaction with the ' SNCRID
All of the 70 NCR substitutions we identified in our genetic screen disrupted the
70 NCR/' SNCRID interaction in the bacterial two-hybrid assay, with the exception of
70 substitution A370V. Furthermore, this substitution did not significantly increase Qmediated antitermination in vitro, and at least some of its in vivo effect may have been
caused by its effect on Q protein levels. Nonetheless, residue 370 lies on the same
surface as the other residues affected by suppressor substitutions, and substitution A370V
had subtle, but reproducible effects on 70-dependent pausing and promoter escape (see
Figs. 4 and 8). To determine whether other substitutions at this position might disrupt the
interaction between the 70 NCD and the ' SNCRID, we introduced mutations specifying
the remaining 18 amino acid changes at 70 position 370 into plasmid pACCI-70 94448 and tested the effects of these substitutions on the 70 NCD/' SNCRID interaction in
the bacterial two-hybrid assay. Several of these substitutions (A370D, A370E, A370G,
A370K, A370N, A370Q, A370R, A370S, A370T) specifically weakened the 70 NCD/'
SNCRID interaction (data not shown) and each of these also suppressed the defect in Qmediated antitermination caused by the 70 L402F substitution in vivo (data not shown).
In addition, two substitutions, A370F and A370Y, specifically strengthened the 70
NCD/' SNCRID interaction (Fig. S2A and S2B). We suspect, therefore, that residue 370
is at or very close to the 70 NCR/' SNCRID interface. Because the 70 NCR and '
SNCRID are likely more constrained in the context of the RNAP holoenzyme than as
tethered protein fragments in the bacterial two-hybrid system, it is possible that the
conservative A to V change may perturb the 70NCR/’NTD interaction in the context of
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holoenzyme without detectably perturbing the interaction between the tethered protein
fragments.
The proposal that the interaction between the 70NCR and the ' SNCRID inhibits
70-dependent pausing and abortive RNA synthesis suggests that 70 substitutions A370F
and A370Y, which strengthen this interaction, should decrease both 70-dependent
pausing and abortive RNA synthesis. As a consequence of decreased pausing, these
substitutions should also impair Q antitermination function. To test these predictions,
we purified 70 A370F and 70 A370Y and assayed the corresponding reconstituted
RNAP holoenzymes in vitro. We examined the effects of these substitutions in the
context of otherwise wild-type 70 because L402F reduces pausing and abortive
transcript synthesis (see Figs. 4 and 8) to such low levels that it would be difficult to
measure further decreases. We found that substitutions A370F and A370Y reduced
terminator readthrough to 22% and 25%, respectively, in the presence of Q, compared
to 35% for RNAP reconstituted with wild-type 70 (data not shown). We then used
E70A370F to assess the effect of strengthening the 70 NCD/' SNCRID interaction on
70-dependent pausing and abortive RNA synthesis. We found that the A370F
substitution reduced the pause capture value at PR’ to 55%, compared to 71% for E70
WT (Fig. S2C) and also that it modestly, but reproducibly, reduced the abortive
probabilities at +12 and +13 (Fig. S2D). These findings thus support the proposal that
the 70 NCD/' SNCRID interaction inhibits 70-dependent pausing and facilitates
promoter escape.
The 70 NCR is important for growth in vivo
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To assess the requirement of the 70 NCR for growth in E. coli cells, we
introduced plasmids encoding mutant 70 proteins into 70 shut-off strain CAG20153, in
which the chromosomal rpoD gene is under control of the trp promoter (Lonetto et al,
1998). Under repressing conditions (in tryptophan-replete medium), expression of rpoD
is greatly reduced, and growth is severely compromised. Tryptophan repression can be
relieved by addition of indole-3-acrylic acid (IAA) to the growth medium. We tested the
abilities of two mutant 70 proteins to complement the lack of wild-type 70 protein in
CAG20153 cells under repressing conditions: 70/A NCR, in which the NCR of E. coli
70 was replaced with the NCR of T. aquaticus (Taq) A, and 70 NCR, in which the
NCR of E. coli 70 was replaced with a seven-residue linker that connects conserved
regions 1.2 and 2.1 of E. coli 38 (see Supplemental methods). Note that two-hybrid
analysis revealed that the Taq A NCR (tested in the context of a A fragment
corresponding to the 70 94-448 fragment) did not interact detectably with the E. coli '
55-261 fragment (ML and AH, unpublished).
Plasmids directing the synthesis of wild-type 70, 70/A NCR, or 70 NCR were
transformed into strain CAG20153 cells and transformants were selected on LB medium
supplemented with IAA and the appropriate antibiotics. Transformants were then restreaked onto LB medium lacking IAA. Cells directing the synthesis of plasmid-encoded
70/A NCR or 70 NCR failed to form colonies or formed tiny colonies, respectively,
under repressing conditions compared to cells directing the synthesis of plasmid-encoded
wild-type 70 (Fig. S3A). We next examined the ability of 70 NCR to support cell
growth in liquid culture. CAG20153 cells containing plasmids directing the synthesis of
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wild-type 70, 70 NCR, or no 70 were grown to stationary phase in LB + IAA and then
diluted into LB – IAA. The presence of 70 NCR permitted only slightly more growth
than the vector control, whereas plasmid-encoded wild-type 70 permitted efficient
growth (Fig. S3B). The slow growth of cells containing no plasmid-encoded 70 can be
attributed to leakiness of the trp promoter, stability of the chromosomally encoded wildtype 70 present before dilution of the cells into medium lacking IAA, and/or residual
IAA from the stationary phase culture. To determine whether the growth defects of cells
directing the synthesis of 70 NCR or 70/A NCR reflect a functional requirement for
the NCR or, instead, reflect defects in synthesis or stability of the mutant proteins, we
performed Western blots of mid-log LB + IAA cultures of CAG20153 cells containing
plasmids directing the synthesis of wild-type 70, 70/A NCR, or 70 NCR. We found
that 70/A NCR and 70 NCR were present at levels comparable to that of
chromosomally encoded wild-type 70 (Fig S3C). We therefore conclude that, although
not absolutely required for cell viability, the 70 NCR is important for normal growth.
To determine whether or not a single amino acid substitution in the 70 NCR that
weakens its interaction with the ' SNCRID would have any effect on cell growth, we
introduced a plasmid directing the synthesis of 70 K299E into strain CAG20153 cells.
Unlike cells containing plasmid-encoded 70/A NCR or 70 NCR, cells containing
plasmid-encoded 70 K299E exhibited no apparent growth defect when plated on LB
medium lacking IAA (data not shown). We suspect that a single amino acid substitution
may not suffice to fully disrupt the 70 NCR/' SNCRID interaction in the context of the
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RNAP holoenzyme. However, we cannot exclude the alternative possibility that the 70
NCR has another functional role that is important for cell growth.
Weakening the interaction between the 70 NCR and the ' SNCRID does not
specifically affect open complex stability
To determine whether the increases in abortive RNA synthesis caused by the
substitutions that disrupt the 70 NCD/' SNCRID interaction are the result of changes in
open complex stability, we measured the kinetics of open complex dissociation for
RNAP reconstituted with wild-type 70, 70 L402F, and the doubly substituted 70
proteins. Open complexes were pre-formed on a PR' promoter fragment extending from 109 to +145 before addition of heparin, and the fraction of heparin-resistant complexes
remaining at each time point was measured by nitrocellulose filter binding (Roe et al,
1984; Ross and Gourse, 2005). Control experiments indicated that formation of heparinresistant complexes was dependent on the PR' –10 element and that heparin prevented the
formation of new open complexes but did not actively dissociate pre-formed open
complexes (data not shown). Fig. S4 shows that the 70 NCR substitutions can decrease
(R281C, R285H, K299E, K371E), increase (E284K, I367F, A370V), or have no
significant effect on (F306S, K359E) open complex half-life. Among the substitutions
that decreased open complex half-life were two that had particularly strong effects on
abortive transcript synthesis (R281C and K299E). Thus, we observed no correlation
(positive or negative) between the effects of our 70 NCR substitutions on open complex
stability, on the one hand, and abortive yields, on the other. We conclude, therefore, that
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the interaction between the 70 NCR and the ' SNCRID affects abortive transcript
synthesis through a mechanism that does not involve open complex stability.
Complementary charge reversal substitutions that partially restore the interaction
between the 70 NCR and the ' SNCRID
Our screen for ' substitutions that suppressed the defect in early elongation
pausing caused by 70 substitution L402F uncovered five charge reversal substitutions
(E148K, E162K, E170K, E171K, and E175K), each of which weakened the interaction of
the ' SNCRID with the 70 NCR as detected in the two-hybrid assay. Among the
suppressor substitutions that we identified in the 70 NCR (i.e. those that suppressed the
effect of substitution L402F on early elongation pausing), three were complementary
charge reversal substitutions: K299E, K359E, and K371E. Each of these 70
substitutions also weakened the 70 NCR/' SNCRID interaction. To explore the
possibility that any of these complementary charge reversal substitutions identify
oppositely charged residues that interact at the 70 NCR/' SNCRID interface, we used the
two hybrid assay to look for mutant-suppressor pairs. That is, we tested all fifteen
possible combinations of 70 and ' substitutions for any that resulted in an increased
interaction between the 70 NCR and the ' SNCRID over that observed when the
substitutions were tested singly. We identified two mutant-suppressor pairs: 70 K371E
in combination with either ' E170K or ' E171K (Fig S5). The suppression was only
partial, but in each case the disruptive effects of the individual substitutions were greater
than the disruptive effect of the two substitutions assayed in combination. We therefore
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suggest that 70 residue K371 closely approaches ' residues E170 and E171 when the
70 NCR interacts with the ' SNCRID.
Supplemental methods
In vivo assay of pausing at the PR'/plac hybrid promoter
Strains. Strain BN343 contains an F' episome bearing the PR’-plac hybrid promoter
fused to a lacZ reporter gene (Nickels et al, 2004). The PR’-plac promoter consists of
sequence extending from –109 to +232 of PR’ with the sequence between –6 and +23
replaced with the corresponding sequence from plac. In strain BN416, the F' episome
carries a derivative of PR’-plac bearing the mutations A(+2)G and T(+6)A (Nickels et al,
2004). Strain ML142 (FW102 with rpoD-K299E linked to a kanamycin resistance gene)
was constructed using the method described in Nickels et al, 2006. Strain BN161 is
FW102 with a kanamycin resistance gene linked to the wild-type rpoD locus (Nickels et
al, 2002). Strain BN105, a derivative of FW102 that does not produce any functional
GreA, carries a greA::CM allele (Nickels et al, 2004). P1 transduction was used to
transfer rpoD-WT-KanR and rpoD-K299E-KanR to strain BN105 to create strains ML26
(FW102 greA::CM rpoD-WT-KanR) and ML156 (FW102 greA::CM rpoD-K299EKanR). Strains BN343 and BN416 were mated with strains ML26 and ML156 to create
the following greA::CM reporter strains: ML157 (rpoD-WT; PR’-plac), ML159 (rpoDK299E; PR’-plac), ML163 (rpoD-WT; PR’-plac A[+2]G ,T[+6]A), and ML164 (rpoDK299E); PR’-plac A[+2]G ,T[+6]A).
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Assays. We examined the ability of Q to stimulate lacZ expression in reporter strain
cells containing either chromosomally encoded wild-type 70 or chromosomally encoded
70 K299E by introducing either plasmid pBRQ or plasmid pBRQ into reporter strains
ML157, ML159, ML163, and ML164. The transformed cells were grown in LB
supplemented with 10 g/mL tetracycline and 100 g/mL carbenicillin. IPTG was
provided at 200 M to induce maximal expression of Q from pBRQ and to prevent the
binding of Lac repressor (encoded by the F' episome) to the lac operator present in the
initially transcribed region of the PR’-plac hybrid promoter. The cells were assayed for
-galactosidase activity as described (Dove and Hochschild, 2004). The fold stimulation
of lacZ expression mediated by Q was determined by dividing the units of galactosidase activity obtained in the presence of Q by those obtained in the absence of
Q.
In vivo assays with 70 proteins lacking the 70 NCR
Plasmids pBR70/A NCR and pBR70 NCR are derivatives of pBR70 in which
sequences encoding residues 123-374 (corresponding to the NCR) were replaced with
sequences encoding residues 121-197 of the Taq A protein or residues 83-89 of the E.
coli 38 protein, respectively. Strain CAG10153 has the chromosomal rpoD gene under
control of the trp promoter and linked to a chloramphenicol resistance gene (Lonetto et
al, 1998). This strain is also recBC, sbc, zgh::Tn10.
Cells were grown in LB supplemented with 10 g/mL tetracycline and 100
g/mL carbenicillin. Nonrepressing medium also contained 0.2 mM indole-3-acrylic
10
acid (IAA). For growth curves, stationary phase LB + IAA cultures were inoculated into
LB – IAA at a 1:100 dilution and growth was assayed by OD600.
The 70 monoclonal antibody used in the Western blotting experiment was
obtained from Neoclone.
Dissociation Kinetics
Open complexes were formed by incubating RNAP (16 nM) with a 32P-labeled
PR' promoter fragment (0.1 nM) for 10 min at 30C in binding buffer (10 mM Tris-Hcl
[pH 8.0], 100 mM KCl, 10 mM MgCl2, 1mM DTT). Heparin was added to 10 g/ml,
and aliquots were removed at various time points, filtered, and the radioactivity
quantified. The promoter fragment extended from –109 to +145 of PR' and was made by
end-labeling an EcoRI-XmnI fragment of pFW11-PR'-lacZ (Nickels et al, 2002). The
fraction of complex remaining at each time point was plotted and fitted to the exponential
equation F = e-kt, where F is the fraction of complexes remaining at time t.
Supplemental References
Dove SL, Hochschild A (2004) A bacterial two-hybrid system based on transcription
activation. Methods Mol Biol 261: 231-46
Lonetto MA, Rhodius V, Lamberg K, Kiley P, Busby S, Gross C (1998) Identification of
a contact site for different transcription activators in region 4 of the Escherichia coli
RNA polymerase 70 subunit. J Mol Biol 284: 1353-1365
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Marr MT, Roberts JW (2000) Function of transcription cleavage factors GreA and GreB
at a regulatory pause site. Mol Cell 6: 1275-1285
Nickels BE, Mukhopadhyay, J, Garrity SJ, Ebright RH, Hochschild A (2004) The 70
subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter. Nat
Struct Mol Biol 11: 544-550
Nickels BE, Roberts CW, Roberts JW, Hochschild A (2006) RNA-mediated
destabilization of the 70 region 4/ flap interaction facilitates engagement of RNA
polymerase Q antiterminator. Mol Cell 24: 457-468
Nickels BE, Roberts CW, Sun H, Roberts JW, Hochschild A (2002) The 70 subunit of
RNA polymerase is contacted by the Q antiterminator during early elongation. Mol Cell
10: 611-622
Roe JH, Burgess RR, Record MT Jr (1984) Kinetics and mechanism of the interaction of
Escherichia coli RNA polymerase with the lambda PR promoter. J Mol Biol 176: 495522
Ross W, Gourse RL (2005) Sequence-independent upstream DNA-CTD interactions
strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association. Proc
Natl Acad Sci USA 102: 291-296
Supplemental Figure Legends
Figure S1. Effects of 70 NCR and  SNCRID substitutions on promoter-proximal
pausing in vitro and in vivo. (A) Shown are representative plots of the percentage of
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elongation complexes paused throughout the transcription time courses with the PR'
template (data for 70 L402F, 70 R281C/L402F, 70 K299E/L402F, and 70
I367F/L402F). The percentage of elongation complexes paused [100 (16-nt +17-nt)/(16nt +17-nt + T)] was approximated at each time point, plotted, and fit to the exponential
equation Y = Y0e-kt, where Y is the percentage of elongation complexes that are paused at
time t and Y0 is the percentage of elongation complexes that initially pause. (B) Shown
are plots of the percentage of elongation complexes paused [100 (17-nt +18-nt)/(17-nt
+18-nt + T)] throughout the transcription time courses with the placUV5 template (data
for a representative trial with 70 WT and 70 K299E). (C) Shown are plots of the
percentage of elongation complexes paused [100 (16-nt +17-nt)/(16-nt +17-nt + T)]
throughout the transcription time courses with the PR' template (data for a representative
trial with ' WT, ' E148K, and ' E175K, assayed in the context of the 70 L402F
holoenzyme). (D) Effect of Q on transcription in vivo from PR'/plac hybrid promoters.
Cells containing the indicated reporter (lacZ fused together with an upstream terminator
to the PR'/plac hybrid promoter bearing either a wild-type or mutated pause element) and
encoding either wild-type 70 or 70 K299E at the chromosomal rpoD locus were
transformed with a plasmid that did or did not encode Q and assayed for -galactosidase
activity. Shown is the fold stimulation of lacZ expression mediated by Q. The cells
were assayed in the presence of 200 M IPTG to induce expression of Q and to prevent
the binding of Lac repressor to the lac operator present in the initially transcribed region
of the PR’-plac hybrid promoter. Shown are the averages of at least three independent
sets of measurements (and standard deviations).
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Figure S2. Strengthening the interaction between the 70 NCR and the  SNCRID
decreases pausing and abortive RNA synthesis. (A) Effects of 70 substitutions A370F
and A370Y on the interaction between the  moiety of the CI-70 94-448 fusion protein
and the ' moiety of the -' 55-261 fusion protein. Strain FW102 F'OL2–62 cells
containing compatible plasmids directing the synthesis of the - 55-261 fusion protein
and the indicated CI-70 94-448 fusion protein were grown in the presence of the
indicated concentrations of IPTG and assayed for -galactosidase activity. (B) Effects of
70 substitutions A370F and A370Y on the interaction between the  moiety of the CI70 94-448 fusion protein and the ' moiety of the -' 262-309 fusion protein. Strain
FW102 F'OL2–62 cells containing compatible plasmids directing the synthesis of the ’ 262-309 fusion protein and the indicated CI-70 94-448 fusion protein were grown in
the presence of the indicated concentrations of IPTG and assayed for -galactosidase
activity. (C) Effect of 70 substitution A370F on pause capture on a  PR template.
Approximate values for pause capture were obtained as described in the Fig. 4 legend.
Error bars represent standard deviations from three separate experiments. (D) Effect of
70 substitution A370F on abortive probabilities at +8, +9, +11, +12, and +13. Abortive
probability values were obtained from single-round in vitro transcription assays
performed with a  PR template as described in the Fig. 8 legend. Error bars represent
standard deviations from at least three separate experiments.
Figure S3. Deletion of the 70 NCR causes severe growth defects. Experiments were
performed in 70 shut-off strain CAG20153, in which the chromosomal rpoD gene is
under control of the indole-3-acrylic acid (IAA) inducible trp promoter. (A) Ability of
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plasmid-encoded 70/A NCR (left panel) and 70 NCR (right panel) proteins to support
cell growth on solid medium under repressing (–IAA) conditions. Colony sizes were
similar to wild-type under non-repressing (+IAA) conditions (not shown). (B) Growth
curves of cells containing plasmids directing the synthesis of wild-type 70, 70 NCR, or
no 70. Indicated times are post-dilution into repressing (–IAA) medium. (C) Western
blot of cells containing plasmids directing the synthesis of wild-type 70, 70/A NCR,
70 NCR, or no 70 under non-repressing (+IAA) conditions. Blot was probed with an
anti-70 monoclonal antibody (Neoclone). Positions of wild-type 70, 70/A NCR, and
70 NCR are indicated. Degradation products of wild-type 70 are also visible.
Figure S4. Weakening the interaction between the 70 NCR and the  SNCRID does not
specifically affect open complex stability. Open complexes were formed on a PR'
promoter fragment with RNAP holoenzymes containing the indicated 70 proteins.
Complexes were challenged with heparin to prevent formation of new complexes, and the
fraction of competitor-resistant complexes remaining at each time point was assayed by
nitrocellulose filter binding. (A) Representative plots of the percentage of heparinresistant open complexes remaining at each time point (data for 70 L402F, 70
R281C/L402F, 70 A370V/L402F, and 70 WT). Half-lives were determined by fitting
the curves to the exponential equation (see Supplemental methods). (B) Open complex
half-lives calculated from dissociation time courses. Shown are the averages of at least
three independent measurements (and standard deviations).
Figure S5. 70 substitution K371E suppresses the effects of  substitutions E170K and
E171K on the strength of the 70 NCR/ SNCRID interaction. Strain FW102 F'OL2–62
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cells containing compatible plasmids directing the synthesis of the indicated CI-70 94448 fusion protein and the indicated - 55-261 fusion protein were grown in the
presence of IPTG (20 M) and assayed for -galactosidase activity. The bar graph shows
the results of three independent measurements (and standard deviations). The solid
horizontal line indicates the basal level of lacZ expression in the absence of an interaction
and the dotted horizontal line indicates the level of lacZ expression when the 70 NCR/
SNCRID interaction is weakened by the 70 K371E substitution. Note that the effects of
the  E170K and E171K substitutions are similar to that of the 70 K371E substitution.
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