The Mechanism of Transcriptional Activation by the Topologically DNA-linked Sliding Clamp of Bacteriophage T4

doi:10.1016/S0022-2836(02)00732-5 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 321, 767–784

The Mechanism of Transcriptional Activation by the Topologically DNA-linked Sliding Clamp of Bacteriophage T4 Scott E. Kolesky*, Mohamed Ouhammouch and E. Peter Geiduschek Division of Biology and Center for Molecular Genetics University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA

Three viral proteins participate directly in transcription of bacteriophage T4 late genes: the s-family protein gp55 provides promoter recognition, gp33 is the co-activator, and gp45 is the activator of transcription; gp33 also represses transcription in the absence of gp45. Transcriptional activation by gp45, the toroidal sliding clamp of the T4 DNA polymerase holoenzyme, requires assembly at primer –template junctions by its clamp loader. The mechanism of transcriptional activation has been analyzed by examining rates of formation of open promoter complexes. The basal gp55-RNA polymerase holoenzyme is only weakly held in its initially formed closed promoter complex, which subsequently opens very slowly. Activation (, 320-fold in this work) increases affinity in the closed complex and accelerates promoter opening. Promoter opening by gp55 is also thermo-irreversible: the T4 late promoter does not open at 0 8C, but once opened at 30 8C remains open upon shift to the lower temperature. At a hybrid promoter for s70 and gp55-holoenzymes, only gp55 confers thermo-irreversibility of promoter opening. Interaction of gp45 with a C-terminal epitope of gp33 is essential for the co-activator function of gp33. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: promoter opening; phage T4; replication – transcription coupling; transcriptional activators; co-activators

Introduction Transcription of the late genes of bacteriophage T4, which constitute approximately 40% of the genome, is initiated at more than 30 promoters consisting simply of a TATA box (with a closely adhered-to consensus sequence: TATAAATA) centered approximately one helical turn upstream of the transcriptional start site. The ability to recognize these promoters is conferred on the host cell’s RNA polymerase core (E) by the T4 gene 55 protein (gp55), a truncated (185 amino acid residues) and highly diverged member of the s70 family of proteins. RNA polymerase consisting only of the core enzyme and gp55 accurately initiates transcription at T4 late promoters.1 Transcription of late genes in wild-type T4 phage-infected Escherichia coli also requires a Abbreviations used: gp33, 45, 55, protein products of genes 33, 45, 55, respectively. E-mail address of the corresponding author: [email protected]

second RNA polymerase-binding protein encoded by T4 gene 33 (gp33) as well as the T4 replication protein gp45, and is very strongly dependent on concurrent replication.2,3 Gene 33, the first gene inferred to be an essential regulator of late gene expression,4 encodes the small (112 amino acid residues) acidic co-activator of late transcription.5,6 gp33 binds to RNA polymerase core (Kd , 25 nM)5 through interactions that do not require its 20 C-terminal amino acid residues.5,7 gp33 is not only essential for gp45-activated transcription, but also strongly inhibits basal (gp45independent) transcription by the E·gp55 RNA polymerase.6,7 The co-activator of T4 middle transcription, AsiA, also exerts a dual effect on transcription: it blocks access of the E·s70 holoenzyme to the 2 35 promoter sites of E. coli and T4 early genes, but is also required for MotA-activated transcription by E·s70 of T4 middle genes (which substitute a MotA-binding site for the 2 35 s70 promoter element).8 – 12 Certain co-activators of eukaryotic transcription effect a similar doublebarreled strategy of enforcing the requirement for an activator by repressing transcription in its

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

768 absence13 – 15 although the specific mechanisms of action differ. gp45 is the sliding clamp of the T4 DNA polymerase. Sliding clamps are ubiquitous components of DNA replication in bacteria, archaea and eukaryotes: gp45 is a structural homolog of the b subunit of E. coli DNA polymerase III holoenzyme and of PCNA, the proliferating cell nuclear antigen.16,17 These proteins are toroids with central holes that accommodate a DNA double helix. Each sliding clamp is loaded onto DNA at an internal nick or gap, or at a double-stranded – single-stranded primer –template junction, by its conjugate clamp loader in an ATP hydrolysisrequiring reaction.18 – 24 PCNA and b are a trimer and dimer of subunits with two and three topologically identical domains, respectively. The linkers between these domains allow b and PCNA rings to adopt the pseudo-6-fold symmetry that is a striking feature of each crystal structure.25,26 Similarly, gp45 is a trimer of subunits with two domains that are topologically identical, but the inter-domain linker of gp45 is relatively short and each monomer is more nearly linear so that, in the crystal structure of gp45, the trimeric ring has only its true 3-fold symmetry.27,28 Loading of gp45 onto DNA by its clamp loader, the gp44 –62 complex, is non-processive: the gp44 – 62 complex, which consists of four molecules of gp44 and one of gp62, attaches to and releases from DNA in each gp45-loading cycle.29 – 31 Once gp45 has been loaded, it moves along the DNA thread by free one-dimensional diffusion. Although gp45 trimer bears a net negative charge at neutral pH, the inner surface of the distorted toroid is positively charged, providing electrostatic stabilization for the topological gp45 – DNA linkage, which may be of some significance for stabilizing the gp45 trimer, as there is clear evidence that the closed structure of gp45 seen in crystals27,28 opens in solution.30,32 The topological linkage of gp45 to DNA is less stable than that of b or PCNA.20,33 It is this instability that is thought to be responsible for the strong coupling of T4 late transcription to concurrent DNA replication in vivo.3 gp45 binds to its ligands, the clamp loader, the DNA polymerase (gp43) and the two phageencoded subunits of the T4 late RNA polymerase holoenzyme, gp55 and gp33, through the same lateral face.19,34,35 Binding to gp43, gp55 and gp33 requires related hydrophobic (and acidic) epitopes (consensus core sequence: (S/T)LDFL(Y/L/F)) that are located at the C termini of each of these three proteins and are solely required for gp45dependent functions: the C-terminal epitope of gp55 is dispensable for basal late transcription, the C-terminal epitope of gp33 is not required for its inhibition of basal transcription, and the C-terminal tail of gp43 is not required for basal, non-processive deoxynucleotide addition to elongating DNA chains.7,36 – 38 Two different sites on gp45 have been proposed for interacting with

Transcriptional Activation by a Sliding Clamp

these C-terminal hydrophobic domains of its ligands. The structure of a co-crystal of the sliding clamp of the T4-related RB69 phage with a C-terminal undecapeptide of the RB69 DNA polymerase shows this peptide located over a hydrophobic patch entirely confined to an individual subunit of the RB69 sliding clamp.28 On the other hand, analysis of the assembly of the gp45 – gp43 DNA polymerase holoenzyme at a primer – template junction and of the structure of the gp45 trimer in solution indicates an open, washer-like structure. The open subunit interface of the trimer serves as the primary attachment site of the C-terminal epitope of gp43, and three potential secondary sites (one for each gp45 protomer) are located on one lateral face.30,39 The mechanism of activation of late transcription by gp45 has several unusual features that arise from its topological (rather than site-specific and physical) DNA linkage. (1) Transcriptional activation by gp45 requires DNA loading by the gp44 – 62 complex. Since these loading sites can be located upstream or downstream of the promoter and at a considerable distance from it, they have the formal properties of enhancers. (2) gp45-loading sites only yield transcriptional activation if they are of the appropriate polarity. This restriction arises because gp45 activates transcription only through interactions with one of its lateral faces, and the orientation of gp45 on DNA is determined by the orientation of the gp44 –62 complex on the DNA-loading site. Once gp45 has been mounted on DNA, its orientation cannot be reversed. (3) gp45 moves from its DNA-loading site to its ultimate location at the upstream end of an activated promoter complex by sliding along DNA. As a consequence, transcriptional activation by gp45 is strictly dependent on the availability of a continuous and unobstructed path along DNA between its DNA-loading site and the promoter.34,40,41 The topological linkage of gp45 to DNA is much less stable than that of the two other analyzed sliding clamps, the E. coli b and human PCNA.20,33 Nevertheless, one would expect the rate of one-dimensional diffusion of any sliding clamp on DNA to be high, and consequently the range of action of gp45 as a transcriptional activator to be large. Activation at a promoter located 3 kb from the gp45 DNA entry site has been observed,40 but may be well below the accessible limit. How the sliding clamp generates activation of T4 late transcription is not yet clear. The ability of gp55 to slide along DNA as a ligand of the sliding clamp suggests a direct role in facilitating promoter location,42 but this step of initiation of transcription may not be rate-limiting. A prior analysis suggests that basal late transcription by E. coli RNA polymerase core and gp55 is characterized by the rapid formation of tightly bound closed promoter complexes that open very slowly.43 If this were the case, the rapid opening of late promoters under conditions of activation by gp45 and gp3337

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Transcriptional Activation by a Sliding Clamp

Figure 1. A T4 late transcription unit and its promoter complexes. (a) Schematic drawing of the principal features of the T4 late transcription unit. The T4 gene 23 promoter, P23, and the rrnB T1 transcriptional terminator define a transcription unit that yields a 111 nt transcript. The 30 recessed (150– 200 nt) and 50 recessed (4 nt) non-transcribed strand, and the enhancer-like gp45loading site are also indicated. (b) Basal and gp45-activated promoter complexes detected by DNase I footprinting of the transcribed strand. Component proteins are indicated above each lane. The T4-modified RNA polymerase core (T4 RNAP E) is ADP-ribosylated in the C-terminal domains of its a subunits, has been stripped of gp33 as well as gp55, and does not contain the T4-encoded RpbA subunit. Footprints of basal (Bas) and gp45activated (enhanced; Enh) promoter complexes are indexed at the side; the bent arrow indicates the start site and direction of transcription. (c) The sequence of the transcribed strand and the extent of footprints. The TATA box recognition sequence of gp55 is lightly shaded and sites of increased DNase I cleavage are underlined. RNA polymerase core: 67 nM for lanes 2 and 4 – 8; 240 nM for lane 3.

would imply, qualitatively, that activation operates primarily by facilitating promoter opening. Analysis of abortive initiation in basal and activated transcription specifies that facilitation of promoter clearance can only play a small part in the overall activation by gp45 and gp33.44 Here, we have turned to a quantitative analysis of the kinetics of open promoter complex formation under conditions of basal and gp45activated transcription. We have also examined the effect of gp33 on RNA polymerase – DNA interaction and have detected a previously unsuspected interaction between the gp45 sliding clamp and the late T4 RNA polymerase holoenzyme. We find that gp45 and gp33 together increase the second order rate constant for formation of open promoter complexes at a T4 late promoter several hundredfold. We also find closed basal promoter complexes

to be extremely unstable. Transcriptional activation and co-activation by gp45 and gp33, respectively, increases the stability of the closed promoter complex and also accelerates its subsequent opening. gp45 also eliminates the inhibitory effect of gp33 on RNA polymerase– DNA binding.

Results A compact T4 late transcription unit The DNA construct shown in Figure 1(a) encompasses the essential features of a late transcription unit that can be activated by the gp45 sliding clamp. Its T4 late promoter, and its transcriptional start site are taken from T4 gene 23 (encoding the major T4 head protein). Placement of the E. coli

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Transcriptional Activation by a Sliding Clamp

Figure 2. Rates of opening of basal and activated T4 late promoter complexes, probed by KMnO4 footprinting. (a) Protein components and times of promoter complex formation at 30 8C are indicated above each lane, and reactive T residues are indicated at the side. Lane 16, DNA only, reacted with KMnO4. Lane 17, DNA only, no KMnO4. (b) Basal promoter opening is kinetically first-order; the entire footprint, its upstream (T 2 12) and downstream (T þ 2) extremes open coordinately. Data are fitted to [RPo] ¼ [RPo]max(1 2 exp[2t/t]) with tbasal ¼ 21.7(^ 2.5) minutes. (c) Very rapid opening of the gp45-activated and gp33-coactivated promoter. The curve drawn through the data is for t ¼ 0.25(^ 0.06) minutes and first-order kinetics. T4-modified RNA polymerase core, 120 nM.

Transcriptional Activation by a Sliding Clamp

771

Figure 3. Only open T4 late promoter complexes are stable to challenge with heparin. (a) Comparison of the rates of formation of basal promoter complexes probed by DNase I ((a), lanes 3 – 8) and KMnO4 ((a), lanes 9 –14) footprinting. Components are indicated above each lane in (a). Lane 1 is the DNase I digestion ladder of a sample without RNA polymerase (E), and lane 2 shows that formation of a footprint requires gp55. A þ G ladders are shown at each side. (b) DNase I and KMnO4 footprints from (a) are quantified in the left-hand panel (t ¼ 7.5(^0.2) minutes) and righthand panel (t ¼ 9.1(^ 0.6) minutes), respectively. T4-modified RNA polymerase core, 240 nM.

rRNA B operon transcriptional terminator T1 defines a transcription unit that yields a 111 nt transcript. Loading of gp45 onto this DNA with the appropriate polarity for transcriptional activation is assured by 150 –200 nt of 50 overhanging single-stranded DNA, generated by 30 ! 50 cleavage with exonuclease III, at the downstream DNA end only. Transcripts reading through the T1 terminator continue into this heterogeneous set of single-stranded template ends. In the absence of other interactions, gp45 loaded at the right end of this DNA (as represented in Figure

1(a)) is expected to slide along it by one-dimensional diffusion and readily unload at the opposite (left) end. Blocking this unloading can generate almost close-packed, steady-state occupancy of extensive stretches of linear DNA with gp45.33 Accordingly, a symmetrized lac operator was inserted near the upstream/left end, but it was found that placing the dimeric (monovalent) R3 Lac repressor at this site diminished instead of increasing the yield of gp45-activated transcription. We surmised that inhibition of activated transcription might be due to over-packing of

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Transcriptional Activation by a Sliding Clamp

Figure 4. The kinetic parameters of basal and gp45-activated promoter opening at 30 8C. The best fit to the data is: tbasal ¼ 1.87 £ 103 [RNAP]21 þ 4.0 minutes (R 2 ¼ 0.84) for basal transcription (B) and tactivated ¼ 5.89[RNAP]21 þ 0.24 minutes (R 2 ¼ 0.90) for activated transcription (X), with [RNAP] designating the concentration of T4-modified RNA polymerase core, saturated with gp55 for basal transcription or with gp55 and gp33 for activated transcription. The inset shows the data for activated transcription with expanded ordinate and compressed abscissa scales. The derived parameters KB (M21) and kf (s21) are listed in Table 1.

DNA with sliding clamps and perhaps exacerbated by the particular location of gp45 trimers that would be imposed by Lac repressor bound relatively close to the promoter (and expected to allow placement of only a short row of gp45 trimers upstream of the transcriptional start site). Thus, this aspect of the transcription template was never exploited in the experiments that are described below. The same DNA construct was used for transcription and for analysis of promoter complexes by DNase I and KMnO4 footprinting of the transcribed DNA strand, labeled at its 30 end , 210 bp upstream of the transcriptional start site (as described in Materials and Methods). Preliminary explorations of reaction conditions led to selection of a Standard Reaction Medium with 200 mM potassium acetate, 10 mM magnesium acetate, 33 mM Tris – acetate (pH 7.8), and 5% polyethylene glycol 3300 as its principal components, and 30 8C as the temperature for the analysis that follows. T4 gp32 was used to cover single-stranded DNA and to facilitate gp45 loading.19,41,45 DNase I footprinting under these reaction conditions showed the expected occupancy of the T4 late promoter by T4-modified E. coli RNA polymerase core (ADP-ribosylated at R265 of the a subunits) in conjunction with the components of T4 late transcription (Figure 1(b)). The basal gp55saturated RNA polymerase holoenzyme generated a shorter footprint with protection that extended at its upstream end only to , bp 2 29, and with

increased sensitivity to DNase I cleavage at basepair 2 30/ 2 32 (lane 3). This footprint was not extended or altered by gp45 in the absence of gp33 (lane 7). Promoter occupancy under basal conditions, in the absence of gp45, was greatly diminished by gp33 (compare lane 4 with lanes 3 and 7). An extension of the footprint at the upstream end only, to , base-pair 2 42, was generated by the combined components of activated transcription (lane 5).34 Omission of gp55 eliminated promoter binding (lanes 2, 6 and 8). Generation of these well-defined footprints was achieved by a brief challenge with heparin before DNase I digestion (Materials and Methods). Footprinting of the basal gp55-holoenzyme (assembled with a sixfold excess of gp55 over the T4-modified RNA polymerase core) without heparin challenge yielded a background of general DNA binding incompatible with quantitative analysis of promoter occupancy. Evidently, gp55 does not contribute the strong inhibition of non-specific DNA binding that is provided by E. coli s70 to its conjugate holoenzyme.46 It was noted that gp33 blocks this non-specific binding as well as promoter-specific binding by E·gp55 and also diminished nonspecific binding to DNA by the T4-modified RNA polymerase core (data not shown). The footprints in Figure 1(b) extend downstream of the transcriptional start (Figure 1(c)). We show below that these heparin-resistant T4 late promoter complexes are open. Promoter opening

Table 1. Kinetic parameters of basal and gp45-activated transcription at 30 8C

Basal Activated Fold-stimulation

ka ¼ KBkf (M21 s21)

KB (M21)

kf (s21)

8.8 £ 103 2.8 £ 106 (320 £ )

2.1 £ 106 4.0 £ 107 (19 £ )

4.2 £ 1023 7.0 £ 1022 (17 £ )

The rate of formation of open T4 late promoter complexes was analyzed by KMnO4 footprinting (Figure 2(a)). As has been shown34, basal and gp45-activated promoter complexes open the same DNA segment. That the entire promoter opens at the same rate was verified by quantifying individual bands of the KMnO4 footprint as a

Transcriptional Activation by a Sliding Clamp

773

Figure 5. Comparisons of basal promoter opening under different reaction conditions and with ADP-ribosylated (T4-modified) as well as unmodified E. coli RNA polymerase core. (a) Rates of opening of the basal promoter complex assembled with 125 nM T4-modified RNA polymerase at 37 8C in Standard Reaction Buffer with 200 mM potassium acetate and 5% polyethylene glycol 3300 (X) and in the reaction buffer of a prior analysis,43 with 50 mM KCl (K). Promoter opening was probed with KMnO4 and quantified for the entire transcription bubble (as in Figure 2); t ¼ 6.1 and 11 minutes for promoter opening in acetatePEG and KCl reaction buffers, respectively. (b) Comparison of promoter opening under standard reaction conditions at 30 8C for basal promoter complexes assembled with unmodified (A) or ADPribosylated (O) RNA polymerase core: t ¼ 8.3(^ 0.4) and 13.0(^ 0.5) minutes, respectively. RNA polymerase cores (240 nM) saturated with gp55 and DNA (2 nM) were incubated in Standard Reaction Buffer at 30 8C for the indicated times. Opening of the P23 promoter was assayed by measuring the yield of P23 ! T1 transcript (cf. Figure 1(a)) in a single round of transcription.

function of time of promoter opening. Data for the entire footprint and for its upstream and downstream ends (T 2 12 and T þ 2, respectively) are compared in Figure 2(b). The time-course for promoter opening by a large excess of basal gp55RNA polymerase holoenzyme fits a single exponential yielding a corresponding pseudo-firstorder rate constant kobs of 7.7 £ 1024 s21 (tbasal ¼ k21 obs ¼ 21.7 minutes) for this experiment. Similar kinetics experiments with the basal transcription system consistently yielded an excellent fit to a single exponential. Under these reaction conditions, gp45-activated promoter opening was extremely fast (Figure 2(a)). The fit of data points to a single exponential was less good, due to a slower component com-

prising 10– 20% of the total signal (Figure 2(c)). The proportion of this slower component relative to the total did not vary systematically with the concentration of RNA polymerase. Its principal effect is to generate a systematic underestimation of kobs in this data treatment. We suspect that the slower component of activated promoter opening may be generated by interference of RNA polymerase – DNA binding, including non-specific binding, with gp45 loading and sliding along DNA. gp45activated promoter opening is so rapid that the 30 seconds duration of probing with KMnO4 is comparatively significant. The indicated time in Figure 2 and for calculating kobs has been taken to be the time of addition of heparin and KMnO4, since inactivation of further promoter opening by

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Transcriptional Activation by a Sliding Clamp

the combination of a large excess of heparin competitor and strong oxidizing agent must be very rapid. Rates of promoter opening, as measured with KMnO4 reactivity, and rates of formation of heparin competition-resistant promoter complexes, as measured by DNase I footprinting, were also compared. As shown in Figure 3, the DNase I and KMnO4 footprints of the basal promoter complex developed at essentially the same rate: quantitative analysis of the upstream and downstream segments of the DNase I footprint showed that the entire pattern of DNA protection also developed coordinately. These two results establish that this DNase I footprint, extending downstream to , base-pair þ 17, characterizes the open promoter complex. If an intermediate-state, stable-but-closed promoter complex exists, comparable with the s70-holoenzyme’s I2 complex at the phage lPR promoter and the intermediate complex at the lac UV5 promoter,47,48 it is rapidly converted at 30 8C to a fully open complex. The kinetic basis of transcriptional activation

Figure 6. T4 late promoter complexes remain open after shift to low temperature. (a) DNase I footprints of the transcribed strand. gp45-activated open promoter complexes were formed at 30 8C for 30 minutes (lane 3) and shifted to 0 8C for 15 or 30 minutes (lanes 4 and 5). Lanes 1 and 2, no-RNAP-holoenzyme controls; samples containing DNA, gp45, gp44/62 complex and gp32, but no RNA polymerase core, gp55 or gp33, were digested at 30 8C (lane 1) or 0 8C (lane 2). Lane 6, control showing that stable gp45-activated promoter complexes did not form when all components were assembled and held continuously at 0 8C for 60 minutes before digestion at 0 8C with DNase I. DNase I digestion for 30 seconds used 1 ng enzyme at 30 8C or 6 ng enzyme at 0 8C. (b) Promoter opening probed in the transcribed strand with KMnO4. Basal open promoter complexes were formed at 30 8C for 60 minutes (lane 3) and shifted to 0 8C for 15 or 30 minutes (lanes 4 and 5, respectively). Lanes 1 and 2, no-RNA polymerase controls; samples containing gp32 but no RNA polymerase core or gp55 were assembled and reacted with KMnO4 at 30 8C (lane 1) or 0 8C (lane 2). Lane 6, control showing that open basal promoter complexes did not form when all components (RNA polymerase core, gp55, gp32) were assembled and held at 0 8C for 60 minutes before reaction with KMnO4 at 0 8C. The same concentration of KMnO4 was used at 30 8C and 0 8C. DNA cleavage was single-hit at both temperatures for the segment extending from the radioactively labeled end to the entire open promoter. T4-modified RNA polymerase, 67 nM.

Rates of promoter opening were analyzed at a range of concentrations of RNA polymerase with results that are summarized in Figure 4 and Table 1. Analysis of the data followed the standard simplified two-parameter model of open complex formation,49 invoking rapid and reversible formation of a closed promoter complex (apparent equilibrium association constant KB) followed by a rate-limiting isomerization process of promoter opening (first-order forward rate constant kf) (Table 1). The derived apparent equilibrium constant KB of 2.1 £ 106 M21 for basal E·gp55holoenzyme indicates a very low affinity of the closed complex compared to well-studied promoter complexes of E. coli RNA polymerase s70 holoenzyme, and a relatively low rate constant for isomerization-promoter opening, and therefore a low value for the second-order association rate constant ka. The corresponding values for gp45activated and gp33-coactivated promoter opening show an , 320-fold increase of ka, with an approximately even partition of the activation between increases in KB and kf. For the reasons specified above, this is an underestimation of activation by gp45. Since the slow component of activated promoter opening is not systematically dependent on RNA polymerase concentration, the underestimation due to this complication partitions comparably between KB and kf. Once they were formed, basal and enhanced open promoter complexes were found to be extremely stable to competition by heparin, diminishing by less than 15– 25% in 60 minutes at 30 8C (data not shown). These findings contrast with the outcome of an analysis of basal T4 late transcription published some years ago, which specified a very tightly bound basal closed promoter complex (KB , 109 M21) and slow promoter opening

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Transcriptional Activation by a Sliding Clamp

Figure 7. Open promoter complexes formed by s70 and gp55-RNA polymerase holoenzymes differ in regard to thermoreversibility. (a) The hybrid s70/gp55 promoter from pRT51034 fuses the P23 late promoter (Figure 1(c)) at basepair 218 to upstream sequence containing a consensus 2 35 site for s70 to constitute a strong promoter for gp55 and s70-RNA polymerase holoenzymes. The sequence of the transcribed strand is shown, promoter elements recognized by s70 and gp55 are shaded, and the transcriptional start site is indicated. (b) Thermoreversibility of promoter opening. Promoter complexes were formed on SK510 DNA for 60 minutes in Standard Reaction Buffer lacking PEG 3300 with 67 nM s70-saturated T4-modified RNA polymerase holoenzyme at 30 8C (lanes 7 – 11), 15 8C (lane 12) or 0 8C (lane 13), with 67 nM gp55-saturated (basal) T4-modified RNA polymerase holoenzyme at 30 8C (lanes 4 and 5) or 0 8C (lane 6). Samples were shifted to 15 or 0 8C for the times shown above the respective lanes (lanes 5 and 8 – 11). Background control samples with RNA polymerase core only were incubated at 30 8C (lane 1), 15 8C (lane 2), or 0 8C (lane 3). Promoter opening was probed with KMnO4 at the final ambient temperature: 30 8C for samples shown in lanes 1, 4 and 7; 15 8C for lanes 2, 8, 9 and 12; 0 8C for lanes 3, 5, 6, 10, 11 and 13.

(kf ¼ 5 £ 1023 s21), comparable with kf for basal transcription in Table 1.43 The prior analysis, which was done with unmodified E. coli RNA polymerase core, in a different reaction medium, and at 37 8C instead of 30 8C, used the less direct but more facile abortive initiation assay described by McClure.49 A direct comparison of promoter opening by KMnO4 footprinting at 30 8C in these two reaction media did not, however, show drastic differences, except for a somewhat greater signal intensity under the conditions of this work (Figure 5(a)). The rate of formation of open and transcriptionally competent promoter complexes by unmodified and ADP-ribosylated RNA polymerase core was also compared in our Standard Reaction Buffer (using the rate of acquisition of capacity for a single round of specifically initiating late transcription as the assay), but only small differences were noted (Figure 5(b)), and the ratio of t for unmodified relative to T4-modified polymerase

was not markedly sensitive to polymerase concentration (0.63(^0.03) for 80–360 nM RNA polymerase). In summary, basal T4 late transcription by its gp55-holoenzyme is characterized by a highly unstable closed promoter complex. Activation of this transcription by the gp45 sliding clamp (with co-activation by gp33) substantially stabilizes this closed complex and substantially relieves the ratelimitation on the subsequent steps of promotercomplex isomerization. For the basal promoter complex, the final process of DNA duplex opening is rapid (Figure 3); it is the isomerization of the initially formed complex, characterized by kf, that is rate-limiting for initiation of late transcription. Opening of the T4 late promoter is not thermoreversible A number of open promoter complexes formed by E. coli s70 and s32 RNA polymerase

776

Transcriptional Activation by a Sliding Clamp

Figure 8 (legend opposite)

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Transcriptional Activation by a Sliding Clamp

holoenzymes re-close upon shift to lower temperatures.50 – 53 In some cases, long-lived intermediates in the pathway to promoter opening can be accumulated and trapped in this way. In an attempt to see such intermediates, basal and gp45activated open promoter complexes were formed at 30 8C and then shifted to 15 8C or 0 8C and probed by DNase I or KMnO4 footprinting. We found, to our surprise, that neither the KMnO4 nor the DNase I footprint of the gp45-activated promoter complex dissipated at 0 8C (Figure 6(a) and data not shown). This was equally a property of the basal open promoter complex (Figure 6(b)) and was independent of the presence of polyethylene glycol in the reaction medium (data not shown). The two special circumstances of this irreversible promoter opening are that it occurs at the T4 late promoter (with only a 2 10 site, and no 2 35 site for binding to a s70 region 4.2 paralog), and that it is generated with gp55, a s family protein lacking a counterpart to s70 region 4.2. To explore these considerations, we turned to a hybrid promoter that can be utilized by s70 and gp55-holoenzyme34 by virtue of placement of a consensus 2 35 site (TTGACA in the non-transcribed DNA strand) 17 bp upstream of the T4 2 10 site TATA box (Figure 7(a)). Open complexes on this hybrid DNA were formed at 30 8C with s70 and gp55RNA polymerase holoenzymes and shifted to lower temperatures, exposing a clear-cut difference between these two holoenzymes (Figure 7(b)): the open s70-holoenzyme’s promoter complex (lane 7) re-closed gradually and almost completely at 15 8C (lanes 8 and 9) to the state of a promoter complex held continuously at 15 8C (lane 12), and re-closed rapidly and completely at 0 8C (lanes 10 and 11). Thus the T4 late TATA box offered no special capacity to s70-holoenzyme for retention of promoter opening at low temperature. The gp55RNA polymerase treated the hybrid promoter as a simple late promoter, and held it open upon temperature downshift (lanes 4 –6).

of transcriptional activation by gp45.37,38 gp33 represses basal transcription, and this repression does not require the C-terminal epitope.7 Nevertheless, extensive incubation with DNA does yield heparin-resistant promoter complexes with various combinations of proteins including C-truncated gp33 and gp55 (Figure 8(a)). The footprints of these complexes share a characteristic feature: an extension to bp 2 33 (lanes 4 –6) differing from the footprints of the basal (lane 2) and the enhanced (lane 3) promoter complexes. This small footprint extension requires gp33, but not its C-terminal epitope, and it requires gp45 (cf. lane 2, and also Figure 1(b), lane 3). These heparin-resistant complexes are open promoter complexes (Figure 8(b)) and, like other T4 late promoter complexes, they are heparinstable (data not shown). Nevertheless, the additional interaction that is reflected in the footprint extensions of Figure 8(a), and which implicates interactions involving gp45 and gp33, does not generate transcriptional activation (Figure 9). When the C termini of gp33 and gp55 are both truncated, residual transcription is at the low level of gp33-repressed basal transcription (Figure 9(b)) and the promoter opens at the very slow rate of gp33-repressed transcription (Figure 9(c)). Interaction with the C terminus of gp55 alone only relieves this repression somewhat, approximately to the level of basal transcription, and falling far short of rapidly initiating fully activated transcription (Figure 9(a)). In the presence of gp55 alone, the sliding clamp generates little or no activation of transcription (Figure 9(a)). Although promoter opening under conditions of basal and gp45activated transcription ultimately reaches the same level (Figures 3 and 9(c)), transcript yields differ approximately 2-fold (Figure 9(a)). This relatively small effect may reflect a difference of efficiency in the transition from abortive initiation to promoter clearance.44

Role of the C-terminal domains of the gp45 ligands in transcriptional activation

Discussion

It is already known that the C termini of gp55 and gp33 are determinants of binding to gp45 and

Detailed kinetic analysis of promoter complex formation and promoter opening, principally at the E. coli lac UV5 and phage lPR promoters,47,48,54

Figure 8. A gp45 interaction with the upstream end of the promoter complex that requires gp33, but not its C-terminal epitope. (a) DNase I footprints of the transcribed strand. Late promoter complexes on SK110-ExoIII DNA (Figure 1(a)) were assembled with the components identified above each lane, 67 nM T4-modified RNA polymerase core, gp32, gp44/62 complex and dATP for 60 minutes at 30 8C. CD8 and CD5 are the C-terminally truncated forms of gp55 and gp33, respectively (i.e. lacking their primary gp45-attachment domains). An A þ G ladder and indexing of the footprint are shown at the left. Footprints of the basal (lane 2) and gp45-activated (lane 3) promoter complexes are to be compared with complexes assembled with gp33 CD5 and/or gp55 CD8 (lanes 4 – 6). The latter lack the footprint extension of the enhanced (gp45-activated) complex (lower bracket at the right side), but differ from the basal complex at the latter’s upstream end (arrowheads at the right side). (b) KMnO4 footprinting. Promoter complexes were assembled as for (a). Samples were split, with one half analyzed under standard conditions (even-number lanes; NTP substrates not added), and the other half analyzed 6.5 minutes after addition of heparin, six minutes after addition of ATP, GTP, CTP and UTP to allow a single cycle of transcription (lanes 3, 5, 7, 9). Lane 1, control, lacking gp55 and gp33.

778

specifies the existence of two significant kinetic intermediates along the reaction pathway: an unstable and competitor-sensitive closed complex forms rapidly; isomerization to a still closed, but more stable and competitor-resistant complex is a relatively slow intermediate step at these promoters; a third step of DNA strand separation generates the open complex. We have examined the mechanism by which the sliding clamp processivity factor of the T4 DNA polymerase activates transcription of the T4 late genes within a simpler framework (Table 1).49 Our analysis specifies KB as the equilibrium association constant of the initial step. A competitor (heparin)-resistant closed basal complex was not detected as an intermediate at 30 8C (Figure 3), because the last step of promoter opening is rapid relative to the anticipated prerequisite isomerization step. Thus, kf is the forward rate constant for that intermediate step of the reaction sequence. T4 late genes are transcribed in the infected cell by an RNA polymerase that is ADP-ribosylated in the C-terminal DNA-binding domain of each a subunit (at R265). The basal late promoter binds this T4-modified E. coli RNA polymerase weakly at 30 8C and opens relatively slowly (Figure 4 and Table 1). Activation by the gp45 sliding clamp and by gp33 increases promoter affinity and relieves a strong limitation on the rate of promoter opening. Together these effects generate an approximately 320-fold increase in the apparent second-order reaction rate constant for promoter opening, KBkf. The limitations of our simple method for following fast, activated promoter opening make this an underestimate, primarily because ignoring a minor slow component of promoter opening generates an underestimation of the pseudo-firstorder rate constant kobs; a relatively long exposure to KMnO4 for footprinting probably is a less significant source of systematic error. Rapidmixing and rapid-quench methods will be required to analyze the kinetic parameters of activated transcription further, and with greater precision. A prior analysis of basal transcription reported tight binding for the closed promoter complex (KB ¼ 1 £ 109 M21).43 In those experiments, the kinetics of promoter opening at 37 8C were determined indirectly by measuring the rate of abortive initiation49 and unmodified E. coli RNA polymerase was used in combination with gp55. Direct measurements of promoter opening by unmodified RNA polymerase under the conditions (that is, the buffer and electrolyte concentrations) of the prior work have not provided any insight into the possible sources of the discrepancy (Figure 5). The very tightly bound closed promoter complexes suggested by the prior analysis might be resistant to a brief challenge by heparin, in which case it should be possible to detect their rapid formation by DNase I footprinting. However, the rate of appearance of heparin-resistant basal promoter complexes, measured with DNase I, and the rate of promoter opening, measured with KMnO4,

Transcriptional Activation by a Sliding Clamp

were not significantly different (Figure 3). Evidently, the rapidly forming initial promoter complex is not resistant to heparin challenge at 30 8C, consistent with relatively weak binding. A relatively unstable initial late promoter complex is also more readily reconciled with involvement of gp55, a s subunit that cannot recognize any 2 35 promoter site because it lacks the corresponding homology segment 4.55,56 With a KB of 109 M21, the closed T4 late promoter complex would have to be as much as two orders of magnitude more stable than s70-holoenzyme – promoter complexes with near-consensus 2 35 and 2 10 DNA-binding sites. The expectation that DNAtethering of gp55 and gp33 by the sliding clamp could contribute to transcriptional activation by facilitating formation of an initial closed promoter complex37 and by stabilizing that complex (Table 1) is also more readily compatible with the existence of a weakly held basal complex than one that is excessively stable. The design of the DNA construct that has been used to analyze transcriptional activation (Figure 1(a)) may appear cumbersome or even suboptimal: the sliding clamp, which activates transcription from the upstream end of the promoter complex, is loaded onto DNA (at its efficient primer – template junction loading site57) downstream of the promoter; as a consequence, RNA polymerase bound to the promoter without gp45 blocks the path along DNA that its own activator must take. On the other hand, once gp45 has been released to DNA by the clamp loader, it can interact directly with gp55 or with gp55·gp33-holoenzyme as it slides along DNA, providing an alternative mode of scanning for promoter sequence.42 gp33 represses late transcription by blocking DNA binding. As a consequence, the gp55 – gp33holoenzyme is generally inefficient in binding to non-specific DNA as well as T4 late promoters. Nevertheless, gp33-repressed transcription still allows slow promoter opening (Figure 9(c)) and yields transcripts (Figure 9(b)). Thus, repression by gp33 evidently does not primarily operate by blocking the transition from abortive initiation to promoter escape. gp45 is more than just the antirepressor of gp33, as it clearly does more than merely restore basal transcription (Figure 9(a) and (b)), but it is essentially ineffective for transcriptional activation in the absence of gp33 (Figure 9(a)), despite its interaction with the gp55-holoenzyme through the C-terminal epitope of gp55.37,38,42 In the absence of the interaction with the homologous epitope of gp33, or in the absence of gp33 entirely, there is no activation of transcription above the basal level (Figure 9(a)); in the absence of both the gp33 and the gp55 C-terminal epitopes, gp45 provides no opposition to repression of basal transcription by gp33 (Figure 9(b) and Sanders et al.37). The structural basis of these actions is illuminated by the already referred-to recent work on the assembly of the processive gp45 – gp43 T4

Transcriptional Activation by a Sliding Clamp

DNA polymerase holoenzyme at a primer – template junction, and on the structures of the gp45 trimer as well as of a complex of the gp45 trimer with the C-terminal peptide of gp43.27,28,30,32,39,58 The principal relevant findings of that work are: (1) the gp45 trimeric ring is not a symmetrical closed structure in solution, but capable of assuming multiple open forms, and the open subunit interface of the gp45 trimer is its principal binding site for the C-tail of the DNA polymerase (gp43); (2) a secondary site for this C-terminal epitope is located on one lateral face of each gp45 monomer. It is speculated that secondary sites might be used transiently for docking the C-end of gp43 during the multi-step process of DNA polymerase holoenzyme assembly at the replication fork. In contrast, activation by gp45 of T4 late promoter open complex formation requires a dual interaction with the T4 late RNA polymerase holoenzyme through the C termini of gp55 and gp33. If the two interaction sites on gp45 are non-identical, as seems likely, the activated promoter complex would have to place gp45 in a unique orientation relative to RNA polymerase, rather than in any of three equivalent orientations. gp55 binds more strongly to gp45 than does gp33, and its C-end perhaps occupies the same subunit interface site as the gp43 C-end,58 but this single interaction does little if anything for transcriptional activity (Figure 9(a)). DNA binding by the E·gp55-holoenzyme is blocked by gp33, but this inhibition is clearly reversed in the gp45-activated (enhanced) transcription complex (Figures 2 and 4). It appears likely that anti-repression and transcriptional activation by gp45 involves the reconfiguration of at least a part of gp33 on the surface of the core enzyme; one can imagine gp45 setting this relocation in motion by pulling on gp33 through its C-tail. In its polymerase-free state in solution, the acidic gp33 (calculated isoelectric point ¼ 4.5) is evidently relatively unstructured (Shao et al.59 and unpublished observation). It will be essential to understand how gp33 interacts with the core subunits of RNA polymerase in its free holoenzyme state and in the gp45-activated promoter complex. Once the gp55-holoenzyme has formed an open T4 late promoter complex at 30 8C, it stays open when cooled rapidly to 0 8C (Figure 6). In contrast, opening of the lPR, lPrm and lac UV5 promoters by E·s70 holoenzyme is thermodynamically disfavored at low temperature.30,32,60,61 Augmenting the T4 late promoter with a s70 2 35 consensus site does not affect its recognition by the gp55-holoenzyme (Figure 7(a)), but allows a direct comparison to be made with s70-holoenzyme. The latter opens this hybrid promoter themoreversibly, opening at 30 8C, closing substantially when cooled to 15 8C and completely at 0 8C (Figure 7(b)). The simplest interpretation of thermo-irreversibility of promoter opening would invoke a combination of two effects: (1) DNA strand separation by the gp55-holoenzyme is thermodynamically favored

779

Figure 9. The residual interaction of gp45 does not generate transcriptional activation. (a) and (b) Single round transcription. Open promoter complexes were formed at 30 8C under conditions of potential activation by gp45 (with 67 nM T4-modified RNA polymerase) on SK110-ExoIII DNA for the indicated times and allowed to undergo a single round of transcription by addition of ATP, GTP, CTP, [a-32P]UTP and heparin. (a) Comparison of gp45-activated (W) and basal transcription (X); effect of withholding gp33 (A) and of substituting gp33 CD5 for gp33 (K). (b) Comparison between basal transcription (X; no gp33 or gp45); basal transcription repressed by gp33 (L no gp45); and transcription with gp45, gp55CD8 and gp33CD5 (O). (c) Promoter complexes were formed as specified for (a) and (b), but probed in the transcribed strand with KMnO4. The timecourse of promoter opening (quantified as shown in the top panel of Figure 2(b), with reactivity at T þ 2, 2 6, 28, 29, 2 10 and 2 12 added together) is compared for basal transcription (X; gp45 and gp33 omitted); basal transcription repressed by gp33 (L; no gp45); and transcription with gp45, gp33CD5 and gp55CD8 (O). Two time-points of promoter opening under conditions of full gp45-activation (W) are included as reference.

780

at 0 8C as well as at 30 8C; (2) the reverse step of isomerization has a high activation energy, so that re-formation of the weakly bound initial complex at 0 8C is very slow on the time-scale of these experiments (Figures 6 and 7). Formation of a separate frozen-in form of the open complex at 0 8C is more complex from a mechanistic point of view, but cannot be excluded61 and merits further investigation. Is the interaction of the 2 35 site of this hybrid promoter with s70 region 4.2 (an interaction that has no counterpart in the T4 late promoter) responsible for this difference between the s70 and gp55holoenzymes? “Extended 2 10” promoters such as gal P1 and the T4 middle promoter PrIIB2 also lack a consensus 2 35 site. To our knowledge, they have not been examined for thermoreversible promoter opening, although opening of the gal P1 promoter by E·s70 at relatively low temperature (5 8C) has been noted.62 These two promoters are regulated by activators (CRP at gal P1 and T4 MotA at PrIIB2) that provide an upstream anchor substituting for direct DNA contact at 2 35. Whether upstream tethering of RNA polymerase to DNA through activator proteins affects temperature-dependence and thermoreversibility of promoter opening by E·s70 or whether these differences are intrinsic properties of the gp55-holoenzyme is currently the subject of further analysis (M. Kamali, personal communication). In this connection, it should also be recalled that deletion of a large, poorly conserved segment of the RNA polymerase b subunit (amino acid residues 186 –433) creates a s70-holoenzyme that can open the strong phage T7A2 early promoter at temperatures as low as 2 20 8C.63

Materials and Methods

Transcriptional Activation by a Sliding Clamp

Ni(NTA)-agarose in the presence of 20 mM imidazole. After washing the column with buffer containing 50 mM Tris – HCl (pH 8), 1 M NaCl, 0.1 mM Na3EDTA, 10 mM b-mercaptoethanol, 5% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml of leupeptin, and 1 mg/ml of pepstatin A, the His-tagged gp33 was eluted in the same buffer containing 200 mM imidazole. The eluted material was exchanged into 40 mM Tris –HCl (pH 8.0), 200 mM NaCl, 1 mM Na3EDTA, 1 mM dithiothreitol, 40% (v/v) glycerol, and judged to be . 95% pure by SDS-PAGE. T4-modified RNA polymerase was purified from E. coli RL916 (generously provided by R. Landick, University of Wisconsin), which contains a chromosomal copy of the rpoC gene (encoding the RNA polymerase b0 subunit) His6-tagged at its C terminus, eight minutes after infection at 30 8C with T4D rpbA 2am(S39) phage68 at a multiplicity of infection of ,8. The culture was rapidly chilled by pouring over 220 8C ice, cells were harvested, quick-frozen and stored at 280 8C. All subsequent steps were performed at 0 – 4 8C. Cells were lysed by a freeze-thaw cycle, followed by digestion with lysozyme (for 30 minutes), addition of 0.2% (w/v) sodium deoxycholate and brief sonication, then centrifuged (45 minutes at 27,000g ). The supernatant was adsorbed to Ni(NTA)-agarose in buffer containing 500 mM NaCl, 10 mM MgCl2, 10 mM imidazole, and the column was washed with buffer containing 50 mM NaHepes (pH 7.6), 150 mM NaCl, 10 mM imidazole, 0.1 mM Na3EDTA, 10 mM b-mercaptoethanol, 0.1% (v/v) Tween20, 2.5% glycerol, 0.5 mM PMSF, 1 mg/ml of leupeptin, and 1 mg/ml of pepstatin A. His6-tagged RNA polymerase was eluted in the same buffer containing 200 mM imidazole. The eluted material was adsorbed to BioRex70 and fractionated over a 150 mM – 700 mM NaCl gradient to remove residual AsiA, s70, gp55 and gp33. The b0 -His6 T4-modified RpbA2 RNA polymerase core, which was judged to be ,95% pure by SDS-PAGE, was dialyzed against storage buffer (40 mM Tris – HCl (pH 8.0), 200 mM NaCl, 40% glycerol, 1 mM Na3EDTA, 1 mM dithiothreitol) and stored at 280 8C at a concentration of 1.2 mg/ml (, 3 mM).

Proteins The purification of gp45, gp44/62 complex, gp32, gp33, gp55CD8, gp33CD5, b0 -His6-tagged and untagged E. coli RNA polymerase core has been described or referenced.5,6,37,64,65 gp55 and gp55CD8, which are overproduced in E. coli as insoluble aggregates, were purified from these inclusion bodies under denaturing conditions,66 stored in 6 M guanidine hydrochloride at concentrations of 380 and 105 mM, respectively, and serially diluted into RNA polymerase core (to a final five- or sixfold molar excess of gp55) for each experiment. His10-s70 was also purified under denaturing conditions67 and renatured on Ni(NTA)-agarose, as specified previously.64 The concentrations of guanidine hydrochloride introduced into reaction mixtures in this way were always less than 33 mM. Accordingly, the guanidine hydrochloride concentration was held constant at 33 mM for the kinetic analysis summarized in Figure 4, in which the concentration of RNA polymerase core was varied. N-terminally His6-tagged gp33 was overproduced at 37 8C in E. coli BL21/lDE3 bearing the T7 RNA polymerase-driven expression vector pET21-His6-gp33. Cells were lysed in a freeze – thaw cycle in the presence of 0.2% (w/v) sodium deoxycholate, briefly sonicated in lysis buffer containing 1 M NaCl and centrifuged for 45 minutes at 27,000g . The supernatant was adsorbed to

DNA PCR fragments for transcription and footprinting were lifted out of plasmids placO-SK110-rrnB(T1 þ T2) and placO-SK510-rrnB(T1 þ T2), both derivatives of pTE110.69 placO-SK110-rrnB(T1 þ T2) has a symmetrized lac operator inserted between the EcoRI and BamHI sites of pTE110 (187 – 168 bp upstream of the transcriptional start site of P23), and the E. coli rRNA operon B transcriptional terminators T1 and T2 on an ,225 bp fragment inserted into the DraIII site. The P23 ! T1 T4 late transcription unit yields an 111 nt transcript. To construct placO-SK510-rrnB(T1 þ T2), the 217 bp BamHI-DraIII fragment containing the wild-type P23 T4 late promoter was replaced by a 164 bp fragment from pRT51034 containing the composite gp55 and s70-dependent promoter shown in Figure 7(a). To construct DNA for transcription and footprinting, a 728 bp PCR fragment containing the T4 late transcription cassette in placO-SK110-rrnB(T1 þ T2) was made with an upstream (with respect to the direction of transcription) primer containing a KpnI site and a downstream 50 amino-modified primer. An exonuclease III-resistant upstream 30 overhang was generated by cleavage with Kpn I; 30 -labeled termini (of the transcribed strand) were

781

Transcriptional Activation by a Sliding Clamp

generated with terminal deoxynucleotidyl transferase and [a-32P]ddATP. Further digestion with HindIII generated an ,580 bp end-labeled fragment with a downstream, exonuclease III-sensitive 50 overhang. This fragment was purified on native polyacrylamide gel and quantified by UV spectrophotometry and scintillation counting. Digestion with exonuclease III generated the downstream 150– 200 nt 50 single-stranded overhang that served at the gp45-loading site (and eliminated the T2 terminator). The composite s70-T4 late promoter footprinting probe was made in a similar way from placO-SK510-rrnB(T1 þ T2), but exonuclease III treatment was omitted, as this DNA was only used for forming basal T4 late transcription complexes. DNA for transcription was prepared in the same way, except that 32 P-labeling and associated gel purification were omitted. Formation and analysis of promoter complexes Promoter complexes for transcription and footprinting analysis were assembled in Standard Reaction Buffer (200 mM potassium acetate, 33 mM Tris –acetate (pH 7.8), 10 mM magnesium acetate, 150 mg/ml of bovine serum albumin, 1 mM dithiothreitol, 0.05% (w/v) Brij58, 5% (w/v) polyethylene glycol (PEG) 3300) as follows (concentrations, in parentheses, are specified for the assembled sample in which promoter complexes form): DNA (2 nM) was combined on ice with gp32 (750 nM), gp44/62 complex (160 nM) and dATP (1 mM). When appropriate, a mixture of T4-modified RNA polymerase core enzyme at the specified concentration, His6-gp33, gp55 (each at five- or sixfold molar excess over core enzyme) and gp45 (400 nM of the trimer), premixed on ice, was added, and the sample, in a total volume of 15 ml, was placed at 30 8C to initiate promoter complex formation. For basal transcription, gp33 and gp45 were omitted; for gp33-repressed basal transcription, gp45 was omitted; gp45-activated (enhanced) transcription required all components; gp55 was omitted from control samples examining the core enzyme in the absence of the promoter-recognizing gp55. The presence of specific components is also identified in the individual Figures and Figure legends. For specifically noted experiments, unmodified E. coli RNA polymerase core replaced the T4-modified core enzyme, and C-terminally deleted proteins gp33CD5 and gp55CD8 replaced the respective full-length proteins. At the desired times, samples were taken for analysis and processed as follows.

For KMnO4 footprinting The reaction sample was mixed with 2 ml of a solution containing heparin and KMnO4, yielding final concentrations in the assay sample of 50 mg/ml or 100 mg/ml of heparin and 18 mM KMnO4, respectively, except as specified below. Oxidation was terminated after 30 seconds by adding nine volumes of Stop Mix with 200 mM b-mercaptoethanol. Sample preparation and analysis (precipitation with ethanol, resuspension in 10% (v/v) piperidine) 1 mM EDTA, heating at 90 8C, extraction with phenol/chloroform/isoamyl alcohol, DNA-precipitation, electrophoretic resolution, and visualization by phosphoimaging followed standard procedure. For quantification of data presented in Figures 2 – 5, footprints of the transcription bubble were normalized to invariant background signals upstream of the promoter region. For quantification of data in Figure 9(c), equal quantities of radioactivity were loaded onto each lane of the gel. For single-round transcription Promoter complexes in 15 ml volume were added to 5 ml of ATP, GTP, CTP and [a-32P]UTP (final concentrations 1 mM, 1 mM, 100 mM and 100 mM), heparin (final concentration 100 mg/ml) and 4.5 units of RNAguard in standard reaction buffer for transcription at 30 8C, terminated after seven minutes with nine volumes of Stop Mix (40 mM Tris – HCl (pH 8.0), 20 mM Na3EDTA, 0.4% SDS, 250 mM NaCl, 250 mg/ml yeast tRNA). Addition of recovery marker DNA, RNA precipitation, resolution, analysis on denaturing polyacrylamide gel and quantification of the P23 ! T1 T4 late transcript by phosphoimage analysis followed previously described procedure.64 The following variations of components and procedure are specific to some of the presented experiments: (1) gp44/62 complex was omitted for the basal transcription shown in Figure 5(b). (2) For the comparison of Standard Reaction Buffer with a previously used reaction medium,43 the latter contained 50 mM KCl, 40 mM Tris – HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, and the analysis was done at 37 8C. (3) Because fully duplex SK510 DNA was used to compare the thermoreversibility of basal gp55-dependent and s70-dependent promoter opening (Figure 7), gp32, gp44/62 complex, gp45 and gp33 were omitted. (4) KMnO4 (10 mM) was used for footprints shown in Figure 9(c). (5) Heparin was omitted for the footprints shown in Figures 6(a) and 7(b).

For DNase I footprinting A 2 ml aliquot of heparin (50 mg/ml or 100 mg/ml final concentration) was added, followed 45 seconds later by 1 ng DNase I (in 40 mM Tris – HCl (pH 8.0), 7 mM MgCl2, 100 mM NaCl, 0.1 mM dithiothreitol, 5% glycerol, 5 mM CaCl2, 100 mg/ml BSA) for 30 seconds. Nuclease action was stopped by adding nine volumes of Stop Mix (40 mM Tris – HCl (pH 8.0), 20 mM Na3EDTA, 0.4% (w/v) SDS, 250 mM NaCl, 250 mg/ml yeast tRNA). Sample preparation (extraction with phenol/chloroform/isoamyl alcohol, precipitation with ethanol, resuspension in formamide sample buffer), resolution by electrophoresis on denaturing 6% polyacrylamide gel, and visualization on phosphor image plates followed standard procedure. Fractional protection from DNase cleavage was quantified between base-pairs 2 23 and þ16 and normalized to upstream flanking DNA.

Acknowledgments We are grateful to G. A. Kassavetis, M. Kamali and K. Wong for helpful discussions and advice, to M. Kamali for a careful review of the text, and to the NIGMS for support of this research.

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Edited by M. Gottesman (Received 29 April 2002; received in revised form 27 June 2002; accepted 11 July 2002)