JBC Avanti Polar Lipids

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M707371200 on October 5, 2007

J. Biol. Chem., Vol. 282, Issue 49, 35482-35490, December 7, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/49/35482    most recent
M707371200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grünberg, S.
Right arrow Articles by Thomm, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grünberg, S.
Right arrow Articles by Thomm, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Transcription Factor E Is a Part of Transcription Elongation Complexes*Formula

Sebastian Grünberg{ddagger}, Michael S. Bartlett§1, Souad Naji{ddagger}2, and Michael Thomm{ddagger}3

From the {ddagger}Lehrstuhl für Mikrobiologie, Universitaet Regensburg, 93053 Regensburg, Germany and the §Department of Biology, Portland State University, Portland, Oregon 97207

Received for publication, September 4, 2007 , and in revised form, October 4, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A homologue of the N-terminal domain of the {alpha} subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions –9 and –11. In complexes stalled at +20, TFE cross-linked specifically to positions +9, +11, and +16 of the non-template strand. Analyses of transcription complexes stalled at position +20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The archaeal transcriptional machinery is a simplified version of the polymerase II (polII)4 machinery. For basal in vitro transcription of most promoters, two transcription factors TBP and TFB are sufficient, orthologues to the eukaryotic transcription factors TBP and TFIIB (reviewed in Refs. 1–3). Archaeal genomes encode a protein with a sequence similar to the N-terminal part of the {alpha} subunit of TFIIE. The N-terminal part of TFIIE-{alpha} is sufficient for basal transcription in the polII system (4). This finding suggests that the archaeal version of TFIIE is much like the evolutionary precursor of TFIIE possessing the core functions of this transcription factor.

TFIIE is a general RNA polII transcription factor that stabilizes the preinitiation complex (PIC) in concert with TFIIH (reviewed by Ref. 5). TFIIE binding to polII recruits TFIIH to the preinitiation complex (4, 6, 7). It binds to single-stranded DNA (8) and is required for ATP-dependent promoter opening by the helicase activity of TFIIH. Negative supercoiling of the template and short mismatched heteroduplex DNA around the initiation sites in topologically relaxed templates bypass the requirement for ATP, TFIIH, and TFIIE, indicating an important role of TFIIE and TFIIH in open complex formation (7). TFIIE stimulates both the helicase and the kinase activity of TFIIH (8) responsible for open complex formation and phosphorylation of the C-terminal domain of polII. TFIIE, TFIIH, and ATP play an essential role in promoter clearance even on negatively supercoiled templates (7, 10). Thus, TFIIE, which is released before position +10 from early elongation complexes (11), also plays an essential role in the transition from initiation to elongation.

TFIIE in human cells consist of two subunits {alpha} (57 kDa) and β (34 kDa) that form an {alpha}2 β2 heterotetramer (9, 12). Photochemical cross-linking results revealed interactions of both subunits of TFIIE with promoter DNA in and immediately downstream of the region of the open complex (13) and in another study upstream of the transcription start site (14). Recently, the TFIIE binding site has been located at the Rpb1 clamp domain in the PIC (15).

Open complex formation in the archaeal PIC occurs independent of TFIIH that is not encoded in archaeal genomes. Therefore, analysis of the core functions of TFE is not complicated by interactions with the complex multisubunit factor TFIIH in the archaeal system. Archaeal TFE is not essential for in vitro transcription but stimulates the initiation rate at some weakly expressed promoters (16), and mutagenesis of the TATA box or low concentrations of TBP also make the transcription of strong promoters dependent upon the presence of TFE (17). Little is known on the mechanism by which TFE stimulates transcription. TFE can compensate for TFB mutants in promoter-directed transcription assays and stabilizes PICs containing TFB mutants in electrophoretic mobility shift assays (18). TFE stimulated RNAP recruitment to heteroduplex templates not bound efficiently by TBP-TFB-RNAP complexes alone (18). This finding led to the speculation that TFE stabilizes PICs by enhancing DNA melting and DNA loading. A direct stimulatory effect of TFE on open complex formation, in particular in the upstream part of the bubble, has been shown recently by permanganate footprinting (19). The stimulatory effect of TFE on transcription depends upon the presence of the E'F heterodimer corresponding to polII subunits Rpb7/4 (20), and the interaction of TFE with RNAP is mediated mostly by subunit E' (19). Here, we demonstrate that TFE induces a higher resumption efficiency of stalled elongation complexes and show the presence and position of TFE in initiation and elongation complexes by photochemical cross-linking. The findings reported here suggest an unexpected role of TFE in the stabilization of elongation complexes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Immobilized in Vitro Transcription Assays of Paused Ternary Complexes—C-minus cassettes of the gdh promoter, biotinylated at the 5'-end of the template DNA strand, attached to streptavidin-coated magnetic beads, were used as DNA templates for pausing of early elongation complexes at position +20 relative to the transcription start site +1. Standard reaction conditions were used as described in Ref. 19 in a reaction volume of 35 µl. 168 fmol of immobilized template DNA were incubated for 30 min at 70 °C in 40 mM NaHEPES, pH 7.3, 250 mM NaCl, 5 mM β-mercaptoethanol, 0.1 mM EDTA, 2.5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 40 µM ATP and GTP, 2 µM UTP, 0.15 MBq of [{alpha}-32P]UTP (110 TBq/mmol), 204 nM TBP, 42 nM TFB, and 45 nM reconstituted RNAP (recRNAP) or 23 nM endogenous RNAP (endRNAP). When indicated, endRNAP or recRNAP were replaced by 50 nM RNAP{Delta}E'F (19). TFE or RNAP subunits E'F were added at various amounts as specified in the legend for Fig. 5. The immobilized templates were purified as described (21) and resuspended in 35 µlof transcription buffer preheated to 70 °C. The reactions were split in 2 x 15.5 µl; after volume compensation to 20 µl with preheated (70 °C) transcription buffer, one half was directly denatured for 3 min at 95 °C with loading dye (21). The volume of the second half was compensated to 20 µl with preheated transcription buffer containing 40 µM all NTPs. The reactions were incubated for 3 min at 70 °C and stopped by the addition of loading dye followed by denaturation. Reactions were analyzed by electrophoresis in 28% urea-polyacrylamide gels. Abortive transcripts from immobilized templates were isolated from the supernatant of purified ternary transcription complexes as described (21).

KMnO4 Footprinting of Initiation and Elongation Complexes—The DNA templates used in footprinting experiments resembled the templates used for immobilized in vitro transcription assays but additionally were labeled with [{gamma}-32P]ATP on the free 5'-end of the non-template DNA strand as described previously (21). Footprinting reaction conditions essentially equaled the conditions described in Ref. 19. 70 nM RNAP, 286 nM TBP, 59 nM TFB, 10 µM ATP, GTP, 1.5 µM UTP, and 500 nM TFE (when indicated) were incubated for 5 min at 70 °C in KMnO4 transcription buffer omitting [{alpha}-32P]UTP, bovine serum albumin, and β-mercaptoethanol. Complexes were isolated (21) and washed with 50 µl of KMnO4 transcription buffer (70 °C). Pellets were resuspended in either 25 µlof KMnO4 transcription buffer (70 °C) or 25 µl of KMnO4 transcription buffer (70 °C) containing 500 nM TFE as specified. All reactions were incubated at 70 °C for 2 min. Then, KMnO4 was added to a final concentration of 23 nM. The samples were incubated for 5 min at 70 °C, stopped, and exposed to piperidine treatment as described previously (21).

Photochemical Protein-DNA Cross-linking of Paused Transcription Complexes—Paused elongation complexes were assembled as described (19). DNA templates used in photochemical cross-linking experiments were internally {alpha}-32P-labeled C-minus+20 cassettes of the gdh promoter DNA containing azidophenacylated phosphorothioate at specific locations, prepared and radiolabeled by DNA polymerase-directed incorporation of one radioactive dNTP 3' to the phosphorothioate, essentially as described (22). 2 nM template DNA were incubated with 20 nM TBP, 60 nM TFB, 46 nM RNAP, and 140 or 500 nM TFE as specified in 12.5 µl of transcription buffer containing 40 mM NaHEPES, pH 7.3, 250 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 600 ng of nonspecific competitor DNA (herring sperm DNA), and 40 µM ATP, GTP, and UTP. NTP-C mix was omitted when indicated. Complexes were formed at 70 °C; specific competitor DNA (gdh promoter DNA bp –164 to +113 at 400 nM) was added after 3 min. Reactions were UV-irradiated for 7 min at 70 °C 2 min after the addition of specific competitor DNA. Following UV irradiation, complexes were treated with nucleases and finally analyzed on 4–19% or 8–19% gradient polyacrylamide-SDS gels, essentially as described (23). Radiolabeled proteins were visualized using an image plate and image analyzer (FLA-500, Fuji, Japan). Photochemical protein-DNA cross-linking of initiation complexes experiments were performed as described in Ref. 24, and TFE was added at 140 nM as specified.

Chemical Cleavage of Cross-linked TFE—Ten 12.5-µl transcription initiation complex reactions containing TBP, TFB, TFE, and RNAP were assembled using gdhP derivatized with APB at –9 and radiolabeled at –8 and –7 of the non-transcribed strand, as described above. Following cross-linking, the nuclease-treated reactions were pooled, and proteins were separated by SDS-PAGE 8–24% gradient. The band corresponding to TFE was cut out, and TFE was eluted overnight at 37 °C in 1 ml of 10 mM Tris, pH 8.0, with bovine serum albumin (25 µg/ml). Eluted TFE was concentrated by microfiltration (10,000 molecular weight cut-off), and aliquots were treated with NTCB (50 mM), which cleaves at cysteines, essentially as described (24). Mock treatments were performed under the same conditions but in the absence of NTCB. The 25-µl cleavage reactions were diluted to 500 µl with equilibration buffer (20 mM Tris, pH 8.0, 300 mM NaCl, and 0.01% Tween 20) and concentrated by microfiltration (3,000 molecular weight cut-off) followed by a second addition of 500 µl of equilibration buffer and concentration to ~20 µl. Samples were split, with half representing "input," and half mixed with Ni2+-conjugated paramagnetic beads for 20 min at room temperature. Supernatant was saved ("super"), and the beads were washed two times with 200 µl of equilibration buffer. Protein was eluted from the beads by incubation for 10 min in equilibration buffer plus 250 mM imidazole ("elute"). Input, super, and elute were analyzed by SDS-PAGE 10–24% gradient, and dried gels were visualized by phosphorimaging.

Photochemical Protein-DNA Cross-linking of TFE to Single-stranded DNA—Nonspecific TFE/single-stranded DNA cross-links were performed using six single-stranded DNA oligonucleotides containing the sequence of the non-template strand of the Pyrococcus gdh promoter DNA from –30 to +13. Each oligonucleotide contained an azidophenacylated phosphorothioate at one specific location (at positions –26, –21, –16, –10, –6, –1, and +1 relative to the transcription start site) and was radiolabeled at the 5'-end with [{gamma}-32P]ATP (21). Equal concentrations of radiolabeled and photoreactive oligonucleotides were incubated in binding reactions at a final concentration of 1 µM as described under "Photochemical Protein-DNA Cross-linking of Paused Transcription Complexes" in the absence of NTPs. Templates were incubated with 1.9 µM TFE, rpoF or rpoE', or rpoL at 70 °C for 5 min. Then, the reactions were UV-radiated at 70 °C. After 5 min of further incubation, loading dye was added; the reactions were denatured for 3 min at 95 °C and loaded onto 4–19% gradient polyacrylamide SDS-gels.


Figure 1
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 1.
TFE binds to a mismatched bubble. A, sequence and features of the preopened transcription bubble DNA construct after EcoRI treatment, comprising a mismatch region from position –6 to +6 relative to the transcription start site +1(boxed) with azidophenacylated phosphorothioate at position –3(circled, flash sign) and adjacent radiolabel at position –1(circled, radioactive sign) on the non-transcribed strand. B, RNAP can bind to photoactive preopened bubble DNA in the absence of TBP/TFB. DNA (2 nM) was incubated with various amounts of RNAP (21, 42, 105, and 210 nM) at 70 °C and UV-irradiated before loading for electrophoresis. C, TFE and RNAP bind to the photoactive, preopened DNA template independently. TFE was added at 1.9 µM (lane 2) to photocross-linking reactions. RNAP was added at 105 nM (lane 3), and RNAP and TFE were added at 105 nM and 1.9 µM (lane 4), respectively.

 


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 2.
TFE cross-links to the non-template strand in PICs. A, TFE cross-linking at position –9 occurs only on the non-transcribed strand of the gdh promoter. After transcription complexes were formed with RNAP, transcription factors as specified, and promoter probe photoactivated at position –9 on the non-transcribed strand (left panel) or the transcribed strand (right panel), cross-links were induced by UV irradiation, processed with nuclease, and resolved by SDS-PAGE as described under "Experimental Procedures." Cross-linked proteins were identified by their relative electrophoretic mobilities and indicated at the right side of the gels. B, cross-linking of TFE, TFB, and RNAP in the region from +4 to –21 of the non-template strand of the gdh promoter. Cross-linking to probes specifically photoactivated at the positions indicated in the figure was performed as in A.

 
Photochemical Protein-DNA Cross-linking of TFE and RNAP to DNA within a Preopened Transcription Bubble—For cross-linking of TFE and RNAP to DNA containing a preopened transcription bubble, templates were prepared essentially as described (22) with the following changes. 5' immobilized oligonucleotides containing an phosphorothioate at one specific location were radiolabeled and extended to full template length basically as described in Ref. 22. Then, the two strands were separated via NaOH treatment, and the strand carrying the phosphorothioate with adjacent radiolabel was hybridized to a long oligonucleotide with mismatch region comprising positions –6 to +6 (19) relative to the transcription start site +1 and azidophenacylated as described (22). For native RNAP shift experiments, 2 nM photoreactive DNA template were incubated in the dark with various amounts of RNAP as indicated in the legend for Fig. 1 in transcription buffer at 70 °C for 5 min and UV-irradiated for 7 min at 70 °C. Following electrophoresis in a 5% native gel (22), samples were visualized using an image plate and image analyzer. For cross-linking experiments, templates (and 105 nM RNAP when specified) were incubated at 70 °C. After 3 min, TFE was added to a final concentration of ~1.9 µM. Samples were incubated for an additional 2 min at 70 °C followed by UV radiation, nuclease treatment, and SDS-gel electrophoresis as described under "Photochemical Protein-DNA Cross-linking of Paused Transcription Complexes."


Figure 3
View larger version (60K):
[in this window]
[in a new window]

  		FIGURE 3.
A, NTCB cleavage and purification of cross-linked TFE. –9NT cross-linking reactions containing TBP, TFB, TFE, and RNAP were separated by SDS-PAGE, the TFE band was excised, and the labeled protein was eluted followed by NTCB treatment. Half of the reactions was directly loaded onto an SDS-PAGE gel (lane 2). The other half of the reactions were bound to TALON (Ni2+) beads. Unbound TFE in the supernatant of a binding reaction with Ni2+ beads was analyzed in lane 4, and Ni2+-bound TFE was eluted with imidazole and analyzed in lane 6. In control reactions, the NTCB treatment was omitted (lanes 1, 3, and 5), respectively. B, predicted cleavage profile for N-terminal His-tagged TFE. Since NTCB cuts at cysteine residues, three single hit products are possible, each inducing one long cleavage product (15–17 kDa) containing a winged helix domain and one smaller cleavage product (6–9 kDa) containing the C-terminal region.

 


Figure 4
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 4.
A, TFE stimulates synthesis of short RNA products. The addition of various amounts of TFE to an immobilized transcription assay containing endogenous RNAP (lanes 1–3) leads to a significant increase of synthesis of short RNA products. TFE was added at 36 (lane 2) or 357 nM (lane 3). B, subunits E'F are important for significant stimulation of abortive transcription by TFE. Transcription reactions were conducted as described under "Experimental Procedures" and contained 50 nM RNAP{Delta}E'F. 260 nM E' and 405 nM F were added in lanes 2 and 6, whereas 660 nM E' and 1 µM F were added to the reactions analyzed in lane 3. Increasing amounts of TFE were added as indicated in the figure (71 nM, lane 4; 357 nM, lanes 5 and 6). C, purification of immobilized ternary complexes (21) and of released abortive transcripts analyzed in panels A and B.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TFE Interacts with DNA Upstream of the Open Complex in Preinitiation Complexes—Eucaryotic TFIIE is involved in promoter binding and melting (5), and TFIIE{alpha} is cross-linked with promoter DNA at position –10, immediately upstream of the open complex (25). To investigate the interaction of TFE from Pyrococcus furiosus with the promoter region in the PIC, we conducted photochemical cross-linking assays with single-stranded DNA, a premelted heteroduplex mimicking an open complex, and we also analyzed cross-linking to the gdh promoter at positions upstream of the transcription start site in the PIC.

We used six single-stranded DNA templates extending from position –30 to +4 of the gdh promoter with azidophenacylated phosphorothioate at positions –26, –21, –16, –10, –6, and –1. TFE cross-links to these single-stranded probes were observed after SDS-PAGE (data not shown). In control reactions, no cross-linking was observed with purified RNAP subunits F and L, but subunit E 'was also cross-linked to this probe (data not shown). This finding indicates that archaeal TFE can bind to single-stranded DNA. A heteroduplex with misspaired DNA in the region from +6 to –6 (Fig. 1A) and a cross-linkable derivatization at position –3 was bound by RNAP in electrophoretic mobility shift assays (Fig. 1B) at enzyme to DNA ratios ranging from 20- to 100-fold (lanes 3–5). When binding of TFE, RNAP, and a combination of TFE and RNAP to this template was studied by photochemical cross-linking, TFE, RNAP, and both in combination were found to be cross-linked to position –3 of the non-template strand after SDS-PAGE (Fig. 1C). A probe containing duplex DNA of the gdh promoter derivatized at position –9 was incubated in binding reactions with RNAP, TBP, TFB, and TFE, and cross-linking to TFB and RNAP subunits B, A', and A'' was observed both on the template and on the non-template strand (Fig. 2A, lanes 2–4). By contrast, cross-linking of TFE was only found at the non-template strand (Fig. 2A, lanes 5 in the left and right panel). Cross-linking of TFE to probes photolabeled at the non-template stand between position +4 and –21 was also studied. Strong cross-linking of TFE at position –11 (Fig. 2B, lane 4) and a weak cross-linking signal at position +4 were observed (Fig. 2B, lanes 4 and 8). No cross-linking was observed at positions –21 and –5 (Fig. 2B, lanes 2 and 6). This finding indicates that archaeal TFE interacts specifically with the non-template strand of promoter DNA mainly in the region upstream of or at the upstream end of the transcription bubble. Cross-linking of TFE was only observed in the presence of RNAP and of TBP/TFB (Fig. 2A). This finding indicates that a complete PIC and the recruitment of RNAP to the promoter are a prerequisite for loading of TFE to the PIC.

In the N-terminal domain of TFE from Sulfolobus solfataricus, the crystal structure of a characteristic winged helix-turn-helix motif has been resolved (26; PDB number 191h) that is also conserved in Pyrococcus TFE (see supplemental material). To identify the domain of Pyrococcus TFE responsible for interaction with the non-template strand of the promoter DNA, N-terminal His6-tagged TFE was isolated from gel-purified PICs cross-linked to the non-template strand at position –9 and cleaved by a reagent specific for cysteine residues (NTCB). The cleavage products of TFE consisting of 15.1, 17.3, and 17.6 kDa are expected to contain the N-terminal part of TFE harboring the winged helix-turn-helix motif (Fig. 3B). The purified NTCB cleavage products were incubated with TALON-Ni2+-coated beads, and proteins attached to the beads were purified and eluted from the beads with imidazole. Both the non-bound fraction in the supernatant of binding reactions with Ni2+-coated beads and the fraction eluted with imidazole from the beads were analyzed by SDS-PAGE (Fig. 3A). NTCB-treated cross-linked TFE was cleaved to a major product of ~15 kDa (Fig. 3B, lane 2). This fragment was enriched in the fraction eluted from Ni2+-coated beads (Fig. 3A, lane 6) and not in the supernatant from binding reactions containing the Ni2+-coated beads (Fig. 4A, lane 4). This finding indicates that the N-terminal part of TFE containing the winged helix-turn-helix motif is involved in binding of TFE to DNA in the PIC.


Figure 5
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 5.
A, TFE increases the resumption of stalled elongation complexes. The reaction mixture contained DNA template, TBP, TFB, and 43 nM recRNAP as indicated. TFE was added to 36 nM (reaction analyzed in lanes 3 and 4) or 357 nM (reaction analyzed in lanes 5 and 6). Ternary transcription complexes were stalled at position +20 by the use of a C-minus cassette of the gdh promoter (21) in reactions not containing CTP. Ternary complexes were purified by the use of streptavidin-coated beads, and resumption was analyzed by the addition of a complete set of NTPs (Chase). Transcripts in lanes 2, 4, and 6 represent the fraction of stalled ternary elongation complexes that were unable to resume transcription after the addition of NTPs. The total activity in stalled ternary complexes is shown in the diagram on the right of the gel, and the resumption of stalled complexes is shown in the diagram below the gel. B, subunit E' is essential for the TFE-induced increase of the resumption efficiency of stalled complexes. TFE was added at a molar concentration of 357 nM TFE (lanes 5 and 6), F at 405 nM, and E' at 260 nM as indicated. C, TFE can be recruited only to PICs. Stalled transcription complexes were formed in the absence (lanes 1 and 2 and 5 and 6) and the presence of 500 nM TFE (lanes 3 and 4). Complexes were washed with preheated transcription buffer and resuspended, and one half of the reaction was complemented with a complete set of NTPs (lanes 2, 4, and 6), whereas the other half was directly denatured (lanes 1, 3, and 5). To chase the reactions analyzed in lane 6, TFE was added to purified ternary complexes at a final concentration of 500 nM. Following incubation for 3 min at 70 °C, reactions were denatured and analyzed on 28% urea-PAGE. The total activity in stalled complexes is shown on the right, and the resumption efficiency of stalled complexes is shown below the figure.

 
TFE Stimulates Abortive Transcription—When transcription complexes were stalled at position +20 on immobilized templates containing the gdh promoter (21) and the complexes were purified by magnet particle separation, small amounts of 5-nt abortive transcripts were found in the supernatant of the reaction (see Fig. 4C for the experimental setup) and were therefore released from the complexes (Fig. 4A, lane 1). Abortive transcripts are generally thought to be formed in repeated cycles by promoter-associated RNAP before the enzyme disengages from the promoter (reviewed by 27). The synthesis of abortive transcripts was ~3-fold increased in the presence of TFE (Fig. 4A, lanes 2 and 3). This finding indicates that TFE stabilizes these early promoter-bound transcription complexes. TFE interaction with RNAP has been shown to be dependent upon RNAP subunits E'F (19, 20). Therefore, we studied the effect of TFE on transcription by the reconstituted archaeal core enzyme lacking these subunits (19). As expected, TFE had only a weak stimulatory effect on abortive transcription by the core enzyme, and synthesis of the released 5-nt transcript was significantly increased when the reactions were supplemented with E' and F or subunit E' alone (Fig. 4B, lanes 6 and 7).

TFE Increases the Resumption Efficiency of Stalled Complexes of the Post-escaped State—The archaeal RNAP starts translocation when RNA reaches a length of 10 nt (21) and structural and biochemical studies of ternary polII complexes suggest that ternary complexes reach their full stability when the growing RNA is 14–15 nt in length (28, 29). To investigate the effect of TFE on later steps of transcription, we analyzed the synthesis of a transcript stalled at position +20 and the resumption of stalled transcription complexes in the presence and absence of TFE by the reconstituted Pyrococcus RNAP. TFE had a moderate effect on the formation of stalled ternary complexes (Fig. 5A, compare lanes 1, 3, and 5, and 5C, lanes 1 and 3; a quantification of the results is shown in Fig. 5A in the right panel) consistent with a ~1.5-fold stimulatory effect on the synthesis of run-off transcripts from the gdh promoter (19). We next analyzed the resumption of ternary complexes by the addition of a complete set of NTPs to stalled complexes and studied the decrease of the 20-nt RNA in ternary complexes after the NTP chase. The resumption of stalled complexes formed by the reconstituted RNAP in the absence of TFE was ~50% (Fig. 5A, lane 2). When TFE was added to initiation reactions, it increased resumption from 50 to ~75% (Fig. 5A, compare lanes 2, 4, and 6). Considering that the maximum resumption efficiency is 100%, this finding indicates a significant TFE-induced increase in the resumption of stalled complexes. A quantification of the resumption is shown in Fig. 5A, in the left panel (below the gel). When TFE was not present during initiation reactions but added later to stalled complexes (Fig. 5C, lanes 5 and 6), it did not exert an effect on elongation of stalled complexes. This finding suggests that TFE is recruited only to the PIC and not to stable ternary complexes. To further investigate the role of TFE on the elongation of stalled transcripts, we next purified complexes of the core enzyme stalled at position +20. Consistent with the previously published result (19, 20) that TFE contacts RNAP via E'F, TFE had only a moderate effect (~10%) on resumption of ternary complexes formed by the core enzyme (Fig. 5B, lanes 11 and 12) but dramatically stimulated elongation of stalled RNAP from 10 to ~30 or 35% when E' (Fig. 5B, lanes 5 and 6) and E'F (Fig. 5B, lanes 3 and 4) was added to initial transcription reactions. These findings suggest that TFE added prior to initiation affects the conformation of the elongation complex in the post-escaped state.


Figure 6
View larger version (76K):
[in this window]
[in a new window]

  		FIGURE 6.
TFE stabilizes the transcription bubble in an elongation complex. Lanes 2–4 show a mixed population of open DNA sites in initiation and elongation complexes (paused at position +20 relative to the transcription start site +1 on the immobilized gdh-C20 template). Thymine residues were analyzed by the single strand-specific reagent KMnO4 on a 10% sequencing gel. Reactions containing TBP/TFB and endogenous RNAP (when indicated) were incubated for 5 min at 70 °C, washed, resuspended, and again incubated for 2 min at 70 °C followed by KMnO4 treatment as described under "Experimental Procedures." Reactions shown in lane 3 contained 500 nM TFE added to PICs, whereas in the reactions analyzed in lane 4, TFE was added to stalled ternary complexes. Significantly modified bases in the region of the elongation complex are highlighted with black spots (lane 3). Boxed bands in lanes 2, 3, and 4 were used for quantification of permanganate sensitive signals. The quantification of permanganate footprints in elongation complexes is shown to the right.

 
To investigate the effect of TFE on the resumption of stalled complexes in more detail, we analyzed the permanganate sensitivity of the transcription bubble in both promoter-bound binary complexes and elongated ternary complexes. To this end, the NTP concentration was optimized to obtain mixed populations of promoter-bound and stalled complexes (see "Experimental Procedures"). When TFE was added before the transcription reactions were started with NTPs, both the signals corresponding to the open complex at the promoter and the +11, +14, and +16 signal corresponding to the bubble in elongation complexes were intensified (Fig. 6, compare lanes 2 and 3). This finding indicates that the addition of TFE to initiation reactions also affects the stalled elongation complex. This effect is not due to an increased formation of stalled complexes in the presence of TFE because TFE increased the formation of stalled complexes only by 20% (Fig. 5A), whereas the stimulatory effect on bubble opening in the ternary complex was ~4-fold (Fig. 6, right panel). This finding suggests that TFE increases resumption of stalled complexes by stabilizing the transcription bubble. When TFE was added to reactions containing stalled complexes and these reactions were incubated for a further 2 min, only the modification of thymine residues located in the promoter region was stimulated (Fig. 6, lane 4). This finding confirms our conclusion inferred from analyses of the resumption efficiency of stalled complexes that TFE is unable to bind to stable elongation complexes.

TFE Cross-links in Stalled Elongation Complexes to the Non-template Strand of the Transcription Bubble—Permanganate footprinting revealed that TFE-induced stabilization of the transcription bubble in stalled elongation complexes occurs at positions +11, +14, +15, and +16 (Fig. 6). To investigate the molecular basis for the TFE interaction with elongation complexes in more detail, the immobilized gdh template was photoactivated at positions +9, +11, +16, and +21, both at the template and at the non-template DNA strand and cross-linking of TFE and RNAP to these probes, was studied (Fig. 7). In the panels showing cross-linking of transcriptional components to position +9, +11, and on to the non-template strand of position +21, a control reaction lacking NTPs was done to reveal the cross-linking pattern in preinitiation reactions (right lane in these panels). For each control reaction, no cross-linking of TFE was observed. This finding indicates that the cross-linking signals obtained after the addition of NTP to transcription reactions are specific for stalled elongation complexes. As predicted from published work (22), cross-linking of RNAP subunits B, A', and A'' was observed in the PICs at positions +9, +11, and +16 but not at position +21 (Fig. 7, last lane each in panel, +9, +11, and in the right part of panel, +21). When TFE was added to transcription reactions containing NTPs, TBP and TFB cross-linking of TFE was observed in panels +9, +11, and +16, specifically on the non-template strand (see lanes 6 and 7 in the right part of each panel). No cross-linking of TFE to the non-template strand could be detected at position +21 and on all studied positions of the template strand. Cross-linking of RNAP subunits B and A' was increased in the presence of TFE in some instances (Fig. 7B, panel +9, template strand, and panel +21, non-template strand) and cross-linking of A''decreased by TFE (see panel +11, template strand). Taken together, these findings suggest that TFE affects the conformation of the elongation complex and indicate binding of TFE to the non-template strand in the transcription bubble in elongation complexes.


Figure 7
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 7.
A, sequence and photolabeling of the gdh promoter. The probe used for cross-linking contained bp –39 to +35, relative to the transcription start site, indicated by the bent arrow. The TATA box and the pausing position (Pos.) +20 are boxed, and the locations of photoactive labels (phosphorothioate derivative containing azidophenacyl bromide) on the template and the non-template strand, respectively, are indicated by asterisks. B, stalled transcription complexes were formed with photoactive DNA templates and challenged with nonspecific (nonsp.) competitor DNA and specific promoter DNA (see "Experimental Procedures"). Gdh promoter DNA was added 2 min prior to UV radiation and nuclease treatment (Dnase I and S1 nuclease in combination or Dnase I and S7 nuclease in combination). The addition of specific competitor DNA minimized the amount of free transcriptional components, and therefore, nonspecific cross-linking. The cross-linked proteins were analyzed in 4–19% gradient SDS-PAGE. The position of the azidophenacylated phosphorothioate in each template is indicated at the left side. The left panel shows the resulting cross-links on the template strand, whereas cross-links on the non-template strand are shown in the right panel. Proteins added to each reaction are indicated on the top of the gels. Triangles indicate an increase of TFE from 140 to 500 nM TFE in the reaction. Cross-linked proteins were identified by their relative electrophoretic mobilities and are indicated at the right-hand side of each gel. For control purposes, some reactions were incubated without NTPs (+9T and +9NT, lanes 8; +11T and +11NT, lanes 9; +21NT, lane 7). Cross-linked reactions for position +9T and +21NT were proceeded with S7 nuclease, which can be radiolabeled autocatalytically (the position of the labeled S7 nuclease is indicated). The position of undigested DNA identified in control lane 1, not containing proteins, is indicated. C, schematic diagram of the experimental design for photochemical DNA-protein cross-linking of paused ternary elongation complexes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the first part of this study, we have identified the position of archaeal TFE in the PIC. Archaeal TFE contains a winged helix domain, suggesting a potential for TFE to bind DNA, although the putative DNA recognition helix is negatively charged and binding of this domain to DNA was not detected in electrophoretic mobility shift assays (26). We show here by cross-linking that TFE such as eukaryotic TFIIE (8) can bind to single-stranded DNA (data not shown) and to a preopened bubble both in the presence and in the absence of DNA-bound RNAP (Fig. 1). In addition, TFE is cross-linked specifically to the non-template DNA strand upstream of the transcription start site in PICs (Fig. 2), and cleavage of the cross-linked TFE molecule revealed that the N-terminal domain harboring the winged helix fold is involved in DNA binding (Fig. 3). These data indicate that this domain is involved in interactions of TFE with DNA of the non-template DNA strand in the PIC mainly in the region upstream of the open complex. Intriguingly, TFE has been shown to promote open complex formation in particular in the upstream part of the transcription bubble (19), and the findings reported here suggest that this stabilization of the open complex is mediated by direct interaction of TFE with the non-template DNA strand (Fig. 2). TFIIE{alpha} and TFIIEβ were cleaved by probes in promoter DNA downstream of the TATA box and overlapping the transcription start site in the yeast PIC (30). Cleavage of TFIIE{alpha} mapped to the winged helix domain. The cross-linking data presented here suggest that archaeal and eukaryotic TFIIE{alpha} interact with a similar region of DNA in the PIC as predicted on the basis of the remarkable structural similarity of the winged domain of eukaryotic TFIIE{alpha} and archaeal TFE (26).

Archaeal TFE interacts directly with TBP and RNAP (17). This finding and the absence of TFIIF and of TFIIH in the archaeal transcriptional machinery suggest a major role of TFE as bridging factor between RNAP and TBP (26). The stabilizing effect of TFE on TBP binding and RNAP recruitment may be also responsible for the stimulatory effect of TFE on the synthesis of a 5-nt abortive transcript (Fig. 4). Intriguingly, the synthesis of a 4-nt abortive transcript is a major feature of unstable early transcription complexes in the pre-escape state of polII (reviewed by Ref. 27). The findings reported here and published data (19) support the conclusion that TFE is involved in the synthesis of these abortive transcripts by stabilizing the transcription bubble in promoter-bound complexes in both systems.

Eukaryotic TFIIE has a clearly established role in the TFIIH-driven transition from initiation to elongation (reviewed by Ref. 5), but the release of TFE from early elongation complexes before the formation of a 10-nt transcript was reported (11). Several lines of evidence presented in this report indicate a completely unexpected additional role of archaeal TFE at later stages of elongation. First, TFE enhances the resumption efficiency of transcription complexes stalled at position +20 (Fig. 5). The finding that the addition of TFE to purified stalled complexes has no effect on resumption (Fig. 5c, lanes 5 and 6) indicates that TFE recruitment can occur only during formation of the PIC and not at later stages of the transcription cycle. Second, permanganate footprinting revealed a strong TFE-induced stabilization of the transcription bubble in elongation complexes stalled at position +20 (Fig. 6). This finding suggests that TFE translocates with RNAP following initiation and acts by stabilizing the transcription bubble in elongating complexes. The physical association of TFE with the elongation complex has been clearly shown directly by photochemical cross-linking (Fig. 7). Analysis of immobilized complexes containing RNAP in both the pre-escaped and the post-escaped state revealed that TFE was specifically cross-linked to the non-template strand of the transcription bubble from position +9 to +16 (Fig. 7). The cross-linking pattern of the RNAP subunits A', A'', and B was also modified in TFE containing ternary complexes. These findings provide evidence that TFE is a part of early elongation complexes and also affects the conformation of the ternary elongation complexes. It is tempting to speculate that the presence of TFE in elongation complexes in vivo increases processivity and decreases pause site occupancy. For example, in particular on long transcripts or operons with several genes, the presence of TFE could have a very large cumulative effect. A direct role of eukaryotic TFIIE in elongation has not been shown, but a potential effect on the processivity of polII by modification of subunits has been discussed (reviewed by Ref. 5). Detailed structural studies of elongation complexes stalled in various registers and cross-linking analyses of transcription complexes at later stages of transcription are required for a deeper understanding of the function of TFE in elongating complexes. Our results reveal a novel core function of TFE in elongation that might also be relevant in eukaryotic transcription complexes lacking TFIIH (31).


   FOOTNOTES
 
* This work was supported by grants from the priority program "Genome function and regulation" of the Deutsche Forschungsgemeinschaft (to M. T.) and the American Heart Association Northwest Affiliate to (M. S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

2 Present address: The Scripps Research Institute, Dept. of Cell Biology, IMM-10, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Back

1 To whom correspondence may be addressed: Dept. of Biology, Portland State University, P.O. Box 751, Portland, OR, 97207. Tel.: 503-725-3858; Fax: 503-725-3888; E-mail: micb{at}pdx.edu. 3 To whom correspondence may be addressed: Lehrstuhl für Mikrobiologie, Universitaet Regensburg, 93053 Regensburg, Universitaetsstrasse 31, D-93053 Regensburg, Germany. Tel.: 49-941-943-3160; Fax: 49-941-943-2403; E-mail: michael.thomm{at}biologie.uni-r.de.

4 The abbreviations used are: polII, polymerase II; RNAP, RNA polymerase; recRNAP, reconstituted RNAP; endRNAP, endogenous RNAP; TF, transcription factor; PIC, preinitiation complex; NTCB, 2-nitro-5-thiocyanobenzoic acid; nt, nucleotides; TBP, TATA-binding protein; T, template strand; NT, non-template strand. Back


   ACKNOWLEDGMENTS
 
We thank Jaimie Powell and Michael Micorescu for assistance in cloning and expression of recombinant TFE.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bartlett, M. S. (2005) Curr. Opin. Microbiol. 8, 677–684[Medline] [Order article via Infotrieve]
  2. Geiduschek, E. P., and Ouhammouch, M. (2005) Mol. Microbiol. 56, 1397–1407[CrossRef][Medline] [Order article via Infotrieve]
  3. Thomm, M. (2007) in Archaea Molecular and Cellular Biology (Cavicchioli, R. ed) pp. 139–157, American Society for Microbiology, Washington, DC
  4. Ohkuma, Y., Hashimoto, S., Wang, C. K., Horikoshi, M., and Roeder, R. G. (1995) Mol. Cell. Biol. 15, 4856–4866[Abstract]
  5. Ohkuma, Y. (1997) J. Biochem. 122, 481–489[Abstract/Free Full Text]
  6. Maxon, M. E., Goodrich, J. A., and Tjian, R. (1994) Genes Dev. 8, 515–524[Abstract/Free Full Text]
  7. Holstege, F. C. P., van der Vliet, P. C., and Timmers, H. Th. M. (1996) EMBO J. 15, 1666–1677[Medline] [Order article via Infotrieve]
  8. Kuldell, N. H., and Buratowski, S. (1997) Mol. Cell. Biol. 17, 5288–5298[Abstract]
  9. Ohkuma, Y., and Roeder, R. G. (1994) Nature 368, 160–163[CrossRef][Medline] [Order article via Infotrieve]
  10. Goodrich, J. A., and Tjian, R. (1994) Cell 77, 145–156[CrossRef][Medline] [Order article via Infotrieve]
  11. Zawel, L., Kumar, K. P., and Reinberg, D. (1995) Genes Dev. 9, 1479–1490[Abstract/Free Full Text]
  12. Ohkuma, Y., Sumimoto, H., Horikoshi, M., and Roeder, R. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9163–9167[Abstract/Free Full Text]
  13. Kim, T.-K., Ebright, R. H., and Reinberg, D. (2000) Science 288, 1418–1421[Abstract/Free Full Text]
  14. Robert, F., Forget, D., Li, J., Greenblatt, J., and Coulombe, B. (1996) J. Biol. Chem. 271, 8517–8520[Abstract/Free Full Text]
  15. Chen, H.-T., Warfield, L., and Hahn, S. (2007) Nat. Struct. Mol. Biol. 14, 696–703[CrossRef][Medline] [Order article via Infotrieve]
  16. Hanzelka, B. L., Darcy, T. J., and Reeve, J. N. (2001) J. Bacteriol. 183, 1813–1818[Abstract/Free Full Text]
  17. Bell, S. D., Brinkman, A. B., van der Oost, J., and Jackson, S. P. (2001) EMBO Rep. 21, 133–138[CrossRef]
  18. Werner, F., and Weinzierl, R. O. J. (2005) Mol. Cell. Biol. 25, 8344–8355[Abstract/Free Full Text]
  19. Naji, S., Grünberg, S., and Thomm, M. (2007) J. Biol. Chem. 282, 11047–11057[Abstract/Free Full Text]
  20. Ouhammouch, M., Werner, F., Weinzierl, R. O. J., and Geiduschek, E. P. (2004) J. Biol. Chem. 279, 51719–51721[Abstract/Free Full Text]
  21. Spitalny, P., and Thomm, M. (2003) J. Biol. Chem. 278, 30497–30505[Abstract/Free Full Text]
  22. Bartlett, M. S., Thomm, M., and Geiduschek, E. P. (2000) Nat. Struct. Biol. 7, 782–785[CrossRef][Medline] [Order article via Infotrieve]
  23. Bartholomew, B., Tinker, R. L., Kassavetis, G. A., and Geiduschek, E. P. (1995) Methods Enzymol. 262, 476–494[Medline] [Order article via Infotrieve]
  24. Bartlett, M. S., Thomm, M., and Geiduschek, E. P. (2004) J. Biol. Chem. 279, 5894–5903[Abstract/Free Full Text]
  25. Forget, D., Langelier, M.-F., Thérien, C., Trinh, V., and Coulombe, B. (2004) Mol. Cell. Biol. 24, 1122–1131[Abstract/Free Full Text]
  26. Meinhart, A., Blobel, J., and Cramer, P. (2003) J. Biol. Chem. 278, 48267–48274[Abstract/Free Full Text]
  27. Dvir, A. (2002) Biochim. Biophys. Acta 1577, 208–223[Medline] [Order article via Infotrieve]
  28. Dvir, A., Tan, S., Conaway, J. W., and Conaway, R. C. (1997) J. Biol. Chem. 272, 28175–28178[Abstract/Free Full Text]
  29. Dvir, A., Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9006–9010[Abstract/Free Full Text]
  30. Miller, G., and Hahn, S. (2006) Nat. Struct. Mol. Biol. 13, 603–610[CrossRef][Medline] [Order article via Infotrieve]
  31. Parvin, J. D., and Sharp, P. A. (1993) Cell 73, 53–540

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Bacteriol.Home page
M. Micorescu, S. Grunberg, A. Franke, P. Cramer, M. Thomm, and M. Bartlett
Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus
J. Bacteriol., January 1, 2008; 190(1): 157 - 167.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/49/35482    most recent
M707371200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grünberg, S.
Right arrow Articles by Thomm, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grünberg, S.
Right arrow Articles by Thomm, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals