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J. Biol. Chem., Vol. 278, Issue 33, 30497-30505, August 15, 2003
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From the Universität Kiel, Institut für Allgemeine Mikrobiologie, Am Botanischen Garten 1-9, D-24118 Kiel, Germany
Received for publication, April 8, 2003 , and in revised form, May 26, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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12 nucleotides in all registers. The size of the RNA-DNA hybrid in an
early archaeal elongation complex was estimated between 9 and 12 nucleotides.
For complexes stalled between positions +10 and +20 the size of the
transcription bubble was around 17 nucleotides. This study shows
characteristic mechanistic properties of the archaeal system and also
similarities to prokaryotic RNAP and pol II. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In bacteria, promoter isomerization from closed to open complex catalyzed
by the predominant RNA polymerase holoenzyme
(
'
2
70) occurs
spontaneously in a temperature-dependent manner
(8,
9). By contrast, nuclear RNA
polymerase II (pol II; see Ref.
10) and Escherichia
coli RNA polymerase specific for promoters of genes involved in nitrogen
metabolism ('254; see
Ref. 11) require ATP
hydrolysis for promoter melting. In the pol II system promoter opening
involves the helicase activity of TFIIH
(12). Eukaryotic nuclear RNA
polymerases I and III share with the 70 containing E.
coli RNA polymerase the ability to produce an open complex of 12–15
bp without ATP hydrolysis. In the pol III system the presence of the general
transcription factor TFIIIB is required in addition to open complex formation
(3).
Methods have been described to prepare ternary complexes stalled at different positions. Analyses of these transcription complexes by nuclease and chemical footprinting provided detailed insights into the basic mechanism of initiation of transcription in enteric bacteria and the eukaryotic pol II system. Pol II complexes were subjected to numerous structural alterations during formation of the first 30 phosphodiester bonds (14–16). In bacteria, a discontinuous model of elongation (inch-worming) was inferred from these studies (17, 18). The finding that movement of RNA polymerase along the DNA template was not synchronous with single nucleotide additions was alternatively explained by transient backtracking of RNA polymerase (19). Goldfarb and co-workers (20) provided evidence that the strength of the RNA-DNA hybrid is essential for maintaining stability of transcription complexes by preventing backtracking of RNA polymerase. Irregular footprints observed earlier were interpreted by these authors in the light of their findings as reflections of mixed populations of transcription complexes in productive and backtracked states.
The archaeal transcription system is a simplified version of the eukaryotic
pol II machinery (21,
22). The archaeal TATA box is
recognized by an archaeal TATA box-binding protein (TBP). This interaction is
stabilized by transcription factor B (TFB), a homologue of general pol II
transcription factor TFIIB. This TBP-TFB promoter complex recruits the
archaeal RNA polymerase that shows striking similarity in sequence and subunit
composition to pol II. With the exception of TFE, which is homologous to the
-subunit of pol II transcription factor TFIIE
(23,
24), no other homologues of
the basal eukaryotic transcriptional machinery were detected in archaeal
genomes. Consistent with the lack of TFIIH in Archaea and in contrast to the
striking general similarity to the pol II system, the archaeal RNA polymerase
does not require hydrolysis of ATP for open complex formation at the
tRNAVal promoter of Methanococcus vannielii
(6). We have developed recently
a cell-free transcription system for the hyperthermophile Pyrococcus
furiosus (26). This
highly purified system consisting of bacterially produced TBP and TFB and RNA
polymerase isolated from Pyrococcus cells was used for the
characterization of the archaeal preinitiation complex
(27), analysis of the
trajectory of DNA in an archaeal transcription complex
(28), and first studies on
regulation of transcription in Archaea
(29,
30). Here we used immobilized
templates to purify Pyrococcus ternary transcription complexes
stalled in registers between +5 and +20. Analysis of these complexes by
exonuclease III (exo III) and potassium permanganate (KMnO4)
footprinting provided a detailed view of the early steps of transcription in
Archaea.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-32P]UTP and [
-32P]ATP were purchased
from Hartmann Bioanalytics (Braunschweig, Germany). Templates for in Vitro Transcription and Footprinting Reactions— Nine templates were constructed. All cytosine residues in the non-template strand between the transcription start site and position +20 relative to the transcription start site were substituted by other bases using PCR and the plasmid pUC19 containing the gdh (glutamate dehydrogenase) gene from –95 to +163 from P. furiosus.
The forward primer was complementary to sequences 160 bp upstream of
the transcription start site, and the reverse primer was partly complementary
to sequences from positions –15 to +20 used to induce the point
mutations. After hydrolysis of the amplified fragments with EcoRI,
the resultant DNA fragments contained the promoter and the mutated region
downstream of the transcription start site of the gdh gene. The
fragments were inserted between the EcoRI and SmaI
(compatible to the blunt ends on one side of the fragment produced by PCR
amplification) restriction sites of the vector pUC19. The resulting fragments
were transformed into E. coli JM109. The resulting plasmids
pUC19/gdh-C5, pUC19/gdh-C6, pUC19/gdh-C7,
pUC19/gdh-C8, pUC19/gdh-C9, pUC19/gdh-C10,
pUC19/gdh-C11, pUC19/gdh-C15, and pUC19/gdh-C20
were used to generate transcription templates by PCR of 263–278 bp in
length. Oligonucleotides complementary to DNA sequences 160 bp upstream
and 90 bp downstream of the transcription start site were used as
primers. One primer was labeled with biotin, and the resulting fragments were
attached to streptavidin magnetic beads (Roche Applied Science) according to
the manufacturer's protocol.
Purification of Pyrococcus RNA Polymerase—RNA polymerase from P. furiosus was purified as described previously (26). Expression and Purification of Recombinant Transcription Factors— The transcription factor TBP from Pyrococcus woesei was overproduced in E. coli as described previously (27). The DNA sequences of P. woesei TBP and P. furiosus TBP show 100% identity. TFP from P. furiosus was expressed and purified as described previously for P. woesei (27).
Immobilized in Vitro Transcription Assays—In vitro
transcription assays were performed according to Ref.
26. A standard reaction
mixture (25 µl) contained 60 ng of immobilized template, 660 ng of RNA
polymerase, 150 ng of recombinant TBP, 120 ng of recombinant TFB, 40
µM ATP and GTP, 2 µM UTP, and 0.15 MBq
[-32P]UTP (110 TBq/mmol). The transcription buffer contained
40 mM HEPES, 0.1 mM EDTA, 1 mM
dithiothreitol, 300 mM KCl, and 4 mM MgCl2.
Transcription reactions were performed for 3 min at 70 °C.
To perform footprinting experiments, the immobilized templates were labeled
with [-32P]ATP. [-32P]UTP was not added to
the reaction.
Isolation of Stalled Ternary Complexes—Ternary complexes stalled in in vitro transcription reactions at positions +5 to +11, +15, and +20 relative to the transcription start site were isolated at 20 °C by the use of a magnet, so DNA attached to magnetic beads could be located to one edge of the reaction tube, and the supernatant could be removed. To remove TBP/TFB from promoter DNA, complexes were washed with transcription buffer containing 0.5% N-lauroylsarcosine (NLS) and 40 µM GTP. Then the isolated ternary complexes were resuspended in transcription buffer and either analyzed on a 28% denaturing polyacrylamide gel or supplemented with all four nucleotides (40 µM each) but no additional radioactivity in a total volume of 25 µl. During further incubation for 3 min at 70 °C run-off transcripts were formed. Transcription reactions were stopped by the addition of loading buffer (98% formamide, 10 mM EDTA, and 0.1% each bromphenol blue and xylene cyanol).
Exonuclease III Footprinting—To perform footprinting
experiments, the immobilized DNA templates were labeled with
[-32P]ATP on the free 5'-end of either the coding or
the RNA-like strand, depending on which strand was attached to the magnetic
particle on the 5'-end. The in vitro transcription reaction was
performed as described, but no [-32P]UTP was omitted. After
the complexes had been stalled at positions +5 to +11, +15, and +20 relative
to the transcription start site, they were isolated as described. Then they
were resuspended in 25 µlof reaction buffer for exo III digestion (40
mM KCl, 2 mM MgCl2, 100 mM
Tris-HCl, pH 8.5, and 1 mM dithiothreitol). After addition of 100
units of exo III, the samples were incubated at 37 °C for 15 min. The
reaction was stopped by the addition of loading buffer, and the samples and
sequencing reactions were loaded on a 6% denaturing sequencing gel.
KMnO4 Sensitivity Assay—To
perform KMnO4 probing, the immobilized DNA templates were labeled
with [-32P]ATP on the free 5'-end of either the coding
or the non-coding strand, depending on which strand was attached to the
magnetic particle on the 5'-end. The in vitro transcription
reaction was performed as described, but no [-32P]UTP was
added. After the complexes had been stalled at positions +5to +11, +15, and
+20 relative to the transcription start site, they were isolated as described.
The complexes were resuspended in 25 µl of transcription buffer, and 2.5
µl of potassium permanganate (250 mM) were added. The samples
were incubated for 5 min at 45 °C. The reactions were stopped by the
addition of 1.7 µl of 2-mercaptoethanol and 20 µl of stop mix (1.25% SDS
and 125 mM EDTA). The supernatant was removed, and the modified
immobilized DNA was resuspended in 18 µl of water, and piperidine was added
to a total volume of 20 µl. The DNA was subjected to cleavage by piperidine
for 30 min at 90 °C. Piperidine was removed by ethanol precipitation, and
the dried pellets were resuspended in loading buffer and loaded together with
a sequence ladder onto a 6% denaturing sequencing gel.
To detect the open complex 2.5 µl of KMnO4 (250 mM) were added immediately after incubation of template DNA with TBP, TFB, and RNA polymerase for 3 min at 70 °C, and the reaction was performed for another 3 min at 70 °C. The reaction was stopped and subjected to piperidine treatment as described.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-32P]UTP. After short incubation times between 30 s and 3
min, an RNA product of 20 nt was synthesized as the predominant product (data
not shown). After incubation times between 5 and 45 min, additional products
of 21 and 32 nt probably caused by misincorporation at positions 21 and 22
were observed. Products of the expected size were also transcribed from the
other templates shown in Fig. 1
when transcription reactions were conducted for 3 min
(Fig. 2A).
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Stalled Complexes Are Stable and Transcriptionally Competent—To investigate the stability of stalled ternary complexes, the various biotinylated templates (Fig. 1) were incubated for 3 min in transcription reactions, and ternary complexes were purified by streptavidin-coated magnetic beads. The RNA contained in these purified complexes was analyzed by PAGE. In addition, the RNA released by the RNAP which was not bound to the magnetic beads was analyzed. The ratio of nascent RNA in ternary complexes to released RNA increased with the length of the RNA molecules synthesized (Table I). When isolated complexes were incubated in transcription buffer supplemented with all nucleotides, labeled RNA associated with isolated complexes could no longer be detected (data not shown). This finding suggests that the nascent RNA molecules were retained in functional ternary complexes that were elongated to run-off products after addition of nucleotides. To provide conclusive evidence that the isolated complexes were functionally active, the RNA products released after addition of nucleotides were analyzed. Fig. 2B shows that run-off transcripts were synthesized under these conditions. Therefore, the isolated ternary transcription complexes are still functionally competent and seem suitable for subsequent analyses of footprints of the RNAP and of growth of transcription bubble at each of these stall sites. An additional analysis of the labeled RNA in isolated complexes stalled at each register between +5 and +20 (see Fig. 1) showed that RNA of the expected size was the major product in most cases (Fig. 2A, lanes 9, 11, 13, 15, and 17). Longer exposures of complexes stalled in register +20 and +15 showed the existence of minor RNA products estimated to be 18 and 13 nt in length (Fig. 2A, lanes 15 and 17). At present it is unclear whether these shorter RNA products are caused by pausing of RNAP or whether they are due to hydrolysis of completed RNA from its 3'-end. All complexes stalled between +7 and +20 contained a 5-nt product suggesting the existence of DNA fragments in complex with RNAP paused at position +5. Further analysis of exo III and KMnO4 footprints showed that the movement from +5 to position +6/+7 marks a significant transition in archaeal transcription which is probably a rate-limiting step (see below). However, these complexes stalled at +5 were not arrested because they could be chased after addition of nucleotides (Fig. 2A, lanes 6, 8, 10, 12, 14, 16, and 18).
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Interaction of Stalled RNA Polymerase with DNA Probed by Exonuclease III Footprinting—We used exo III as a probe to identify the upstream and downstream boundaries of RNA polymerase at each of the stall sites. To define the upstream extent of the binding site, linear DNA was 5'-end-labeled with 32P on the template DNA strand, and the biotin label was associated with the 5'-end of the complementary DNA strand. For analysis of the downstream extent of the RNAP-binding site, the 5'-end of the RNA-like strand was labeled with 32P, and the biotin label was attached to the 5'-end of the template DNA strand. Cell-free transcription reactions were conducted at 70 °C and the subsequent purification of transcription complexes at 20 °C. Because exo III was rapidly inactivated at 70 °C (data not shown), ternary complexes were incubated with exo III at 37 °C. At this temperature, initiation of transcription did not occur, but already formed isolated ternary complexes can be elongated by addition of a complete set of nucleotides (data not shown). Therefore, the complexes probed by exo III at 37 °C were transcriptionally active and competent.
When the downstream boundary of the complex stalled at position +5 was analyzed, two distinct signals not present in the control reaction were identified (Fig. 3A, left panel). The strong diffuse band located between positions –19 to –15 corresponds approximately to the downstream end of the TBP/TFB footprint identified in the Methanococcus and Sulfolobus system by DNase I protection analyses (6, 33). The second signal at position +18 corresponds to the downstream edge of RNA polymerase identified in the Methanococcus and Sulfolobus system. After addition of NLS to the complexes, the exo III stall site at position –19/–15 was no longer detected, whereas the second signal at position +18 was not sensitive to NLS treatment (Fig. 3A, left lane in left panel). We therefore conclude that NLS removes TBP/TFB from the template, whereas the archaeal RNA polymerase in ternary complexes remains associated with DNA like eukaryotic pol II (14). Consecutive elongation of RNA from 5 to 8 nt did not cause movement of the downstream edge of RNAP (Fig. 3A and summary of footprinting data in Fig. 5). Between registers +9 and +20 the downstream edge of RNAP translocated approximately synchronously with RNA elongation. The downstream end of the RNAP footprint was located at positions +20, +22, +24 + 26, and +32 in registers +9, +10, +11, +15, and +20 (Fig. 3A and Fig. 5).
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In register +5, a distinct upstream boundary of RNA polymerase could not be identified (Fig. 3B, left lane). The two signals at positions –42 and –35 are almost identical with the upstream edges of the TBP/TFB and TBP DNase I footprint at the Pyrococcus gdh promoter (27). In all archaeal systems investigated, the upstream edge of RNAP could not be directly determined in preinitiation complexes. Addition of RNA polymerase to TBP/TFB promoter complexes caused extension of the protection patterns downstream but not upstream of the TBP/TFB-binding site (6, 33). However, in complexes stalled between position +7 and +9 an upstream edge of RNAP could be identified at position –7 (Fig. 3B and Fig. 5). This finding indicates that a structural transition occurred in the early elongation complex stalled between +7 and +9. The upstream edge of complexes stalled at positions +10, +15, and +20 was located at position –4, +1, and +4 (Fig. 3B and Fig. 5). This finding indicates continuous movement of RNAP with the extension of the RNA chain between registers +10 to +20. To analyze the events during initiation and elongation in more detail, the open region and transcription bubble extension in stalled complexes were analyzed in addition.
Open Complex, Transcription Bubble Progression, and RNA-DNA Hybrid—To investigate open complex formation and transcription bubble extension, we used potassium permanganate (KMnO4) as a probe specific for thymidine (T) residues in single-stranded DNA. To investigate the temperature dependence of open complex formation TBP/TFB was incubated individually or in combination with RNAP with end-labeled DNA fragments containing the gdh promoter ("Experimental Procedures") at 50 and 70 °C. Transcription reactions on linear templates were usually conducted at 70 °C, and as expected T residues in the region of the transcription start site were modified by KMnO4 treatment (Fig. 4, A and B, left panel, and Fig. 5, upper panel) at 70 °C. No KMnO4 footprint was observed at 50 °C or when TBP/TFB alone was incubated at both temperatures with these templates (data not shown). These findings indicate that the RNA polymerase was required for strand separation at the promoter and that the open complex was not formed at 50 °C although ternary complexes can be elongated at temperatures down to 37 °C. Five T residues at positions –6, –4, –2, +2 and +3 were modified on the RNA-like strand (Fig. 4A, left panel), and 2 residues at –7 and –3 were strongly modified on the coding strand (Fig. 4B, left panel). Additional T residues with increased sensitivity to KMnO4 were identified at positions –8 and –9 and at +4 and +5 on the coding DNA strand. These data indicate that the open complex extends from –9 to +5 at the Pyrococcus gdh promoter.
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To investigate progression of the transcription bubble, transcription reactions with the templates shown in Fig. 1 were conducted at 70 °C, the ternary complexes isolated at 20 °C, and the KMnO4 reactivity of T residues in stalled complexes was analyzed at 45 °C. On the RNA-like DNA strand, the modification pattern of the complex stalled at position +5 was basically the same as in the preinitiation complex. On the coding DNA strand T residues at –7 and –3 were modified. The T residue at position +4 showed no sensitivity to KMnO4 (Fig. 4B). This lack of reactivity of T residues close to the 3'-end of nascent RNA was often observed on the coding strand (see Fig. 5 and below) of ternary transcription complexes. We conclude that these T residues are hybridized with nascent RNA and thereby protected from modification with KMnO4. This protection of T residues at the coding strand was used for an estimation of the size of the RNA-DNA hybrid (see below). The finding that the T residues at –8 and –9, in contrast to the T residues at the same position in the open complex, were not sensitive to KMnO4 in register +5 indicates reclosure of the open complex at these positions and movement of the upstream edge of the transcription bubble (see Fig. 5). In complexes stalled at positions +7, +8, +9, and +11, a modified T residue two positions downstream of the stall site was detected on the template strand. By contrast, the T residue 2 nucleotides downstream of the NMP addition site was not modified in complexes stalled at position +6 (see Fig. 4B). This finding indicates that the single-stranded region in the transcription complex can extend beyond the 3'-end of nascent RNA in complexes stalled in registers higher than 6. The complexes stalled at positions +7, +8, and +9 showed very similar reactivity toward KMnO4 on the coding DNA strand (Fig. 4) and most T residues of the RNA-like strand, but the reactivity of the T residue at position –6 was decreased indicating reclosure of the transcription bubble.
A clear change in the modification pattern was observed on the RNA-like strand in registers +10 and +11 (Fig. 4A). The KMnO4 reactivity at positions –6 and –4 was reduced, the reactivity of the T residues at +2 and +3 dramatically increased, and modification of the T residue at position +6 was clearly increased. These findings indicate a major conformational change and movement of the transcription bubble in these registers.
Stalling RNAP at position +15 results in sensitivity of two T residues toward KMnO4 on the RNA-like strand at positions +14 and +11, which were not modified in register +11 (Fig. 4A). In addition, the T residues at positions +6 and +3/+2 were modified. The T residues at positions –4 and –6 showed no reactivity in this complex. On the coding strand in register +15 also strong changes in the modification pattern were detected (Fig. 4B). T residues at positions +4 and +5 showed strong reactivity; the signal at –3 was drastically reduced in intensity, and modification of the T residues at positions –7 and –8 could not be detected (Fig. 4B).
At register +20, again a very significant change of the modification
pattern was observed. On the RNA-like strand, the T residues at positions +16,
+15, +14, and +11 showed reactivity toward KMnO4; in addition, the
T residue at position +6 was sensitive
(Fig. 4A). The T
residues at position +3/+2 showed reduced reactivity. On the coding DNA
strand, the reactivity of T residues at –3 was eliminated; the
sensitivity of T residues at positions +4/+5 was reduced, and a novel T
residue at +8 was modified (Fig.
4B). We estimate the open region at this stall site from
+4 to +20, 16 bp in length. An estimate of the extension of the
transcription bubble in each register of transcription is given in
Fig. 5.
The results presented here allowed also an estimate of the nascent RNA-DNA hybrid length. One striking example is the complex stalled at position +20. On the RNA-like strand, the T residues at positions +11, +14, +15, and +16 were clearly modified (Figs. 4A and 5). Therefore, the T residues on the opposite strand at positions +12, +13, and +19 (see DNA sequence in Fig. 5) must be located within the melted DNA region. However, these T residues on the coding strand showed no KMnO4 signal (Fig. 4B). Considering this finding we suggest that this protection is due to an RNA-DNA hybrid of at least 9 nt. This is a minimal estimate as the next modified T residue on the coding strand which is not protected by hydrogen bonding to adenine in RNA is located at position +8. Therefore, the length of the RNA-DNA hybrid may extend up to 12 nucleotides (indicated by dotted lines in Fig. 5). In such a way, the length of the RNA hybrid was estimated in each register of transcription (summarized in Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Three Different Conformations of RNAP and Two Distinct Structural Transitions Were Observed during Early Steps of Archaeal Elongation—The exo III footprinting data presented here and previous results suggest that the conformation of RNAP does not change during synthesis of the first five nucleotides. The exo III borders of the complex stalled at position +5 analyzed in this study (Fig. 3 and summary in Fig. 5) are basically the same as the limits of the PIC determined in the Pyrococcus and other archaeal systems by DNase I footprinting (6, 22, 27). One striking property shared between the complex stalled at position +5 and the PIC is that an upstream limit of the RNAP-binding site cannot be defined. This finding suggests that the RNAP is in close contact with the transcription factors TPB/TFB assembled around the TATA box/BRE promoter elements (Fig. 5, top) in the PIC and in complexes stalled at position +5. In addition, also the downstream limit of the RNAP-binding site is the same as in the PIC. These findings indicate that the RNAP does not move during synthesis of the first 5 nucleotides.
Our exo III footprinting data indicate that two distinct structural transitions occur between registers +6 and +20. The first transition was observed between registers +6 and +7. The RNAP seems to undergo a conformational change and/or to start translocation indicated by the presence of an RNAP-induced exo III stop signal at position –7 (Fig. 3B and Fig. 5). Beyond register +6 an extension of the transcription bubble 2 nucleotides downstream of the NMP addition site was observed by KMnO4 footprinting (see Figs. 4B and 5; T residues labeled by an asterisk). Thus, two independent methods indicate that a structural transition occurs in complexes stalled at registers +6/+7. The conformation of these complexes is characterized in addition by an unchanged downstream edge of the exo III footprint that is located at position +18. Although we could not detect an upstream boundary of RNAP in register +8 (Figs. 3B and 5) the upstream edge of RNAP was consistently located at position –7 in registers +7 and +9. We therefore assume that the RNAP-binding site in the first transition state extends from positions –7 to +18 over a DNA segment of 25 bp (Fig. 5).
The second clear structural transition occurs in complexes stalled at
positions +10 and +11. Here the downstream part of RNAP starts translocation,
and this movement continues synchronously with RNA elongation up to the stall
position at +20 (Figs.
3A and
5). In each case the distance
between the 3'-end of RNA and the downstream edge of RNAP was 12 bp
(Fig. 5). A somewhat longer but
also constant distance has been found in active eukaryotic
(13) and prokaryotic
transcription complexes (25).
Exo III borders very close to the site of NMP addition are characteristic for
backtracking of RNAP and arrested complexes
(13). Stalling
Pyrococcus RNAP at position +10 produced beside the signal at
position +22 a second exo III pausing site at position +16 that was
significantly closer to the 3'-end of the RNA. This signal at +16 is
likely to be due to backtracking of RNA polymerase. This second complex at
stall site +10 seems not to be arrested because all RNAs isolated in ternary
complexes stalled at +10 could be chased by the addition of NTPs
(Fig. 2A, lane
12, and Fig. 2B,
lane 6). The finding that the distance between the 3'-end of
the transcript to the leading edge of RNAP is constantly 12 bp supports our
former conclusion that all isolated complexes were transcriptionally
competent. The upstream end of complexes stalled between position +10 and +20
could also be clearly identified in each case and move also continuously with
RNA elongation (Figs.
3B and
5). The complexes stalled at
+11, +15, and +20 are characterized by coordinate movement of the active site
and both the leading and trailing edges of RNAP.
Movement of Transcription Bubble and RNA-DNA Hybrid— The conclusions inferred from exo III footprinting were confirmed and extended by analyses of transcription bubble extension in stalled complexes by KMnO4 footprinting. The open region in the PIC was formed in a temperature-dependent manner and extended from position –9 to +5. Considering the limits of the method this is very similar to the open region in the PIC of a tRNAVal promotor of Methanococcus and an rRNA promoter of Sulfolobus which ranged from –11 to –1 and –12 to –1, respectively (6, 7). Up to register +6 no significant movement of the downstream edge of the bubble could be detected (Figs. 4 and 5). Because the three T residues at positions –7 to –9 showed reduced activity toward KMnO4, the process of reclosure of the open region at the upstream end was visible after synthesis of 5 and 6 nucleotides (Figs. 4B and 5). Only weak variations of the KMnO4 sensitivities were observed between registers +7 and +9 (Figs. 4 and 5). The most significant change in these early registers was the extension of the length of the transcription bubble from 12 nt in register +5 to 17 in register +9 (Figs. 4 and 5). A more dramatic change of the KMnO4 sensitivity patterns at positions +10 and +11 indicated a structural transition of the bubble at these stall sites. From analyses of the modifications patterns in complexes stalled at positions +10 + 15 and +20 clearly movements of the bubble at the upstream and downstream edges could be inferred. This finding indicated that in early archaeal elongation complexes both the upstream and downstream edge of the bubble move synchronously with RNA synthesis. In complexes stalled at positions +11, +15, and +20 the open region encompasses 17, 14, and 17 nt in length. Because estimation of the bubble size by KMnO4 footprinting depends on the presence of T residues, the open region indicated by the gray box in Fig. 5 is a lower estimate. We assume that the bubble size is at least 17 nt for complexes stalled between positions +10 and +20.
Analysis of the extent of the open region was complicated by the existence of complexes paused at +5 (visible in the lower part of Fig. 2A) which could cause additional KMnO4 signals in the region of the transcription start site which were not part of the moving transcription bubble. However, careful inspection of the KMnO4 modification patterns allowed clear definition of the major transitions during translocation of the bubble. The formation of a hybrid between the growing RNA chain and the template DNA strand complicated an exact determination of the downstream limit of the bubble at the coding DNA strand. But when the KMnO4 modification patterns on both DNA strands and the weak KMnO4-sensitive signals beyond the site of NMP addition (indicated by an asterisk in Fig. 4B) on the coding DNA strand were considered, it was possible to infer both the extent of the open region and the extension of the RNA-DNA hybrid.
The RNA-DNA hybrid grew continuously with RNA elongation in early registers of transcription. It was at least 2 nt in register +5, 3 in register +6, 4 in register +7, 5 in register +8, 6 in register +9, 7 in register +12, and 8 in register +11 (Fig. 5). When RNAP was stalled at position +15, the length of the RNA-DNA hybrid was at least 8 and at stall site +20 at least 9 nt. The finding that the T residue at position +8 on the coding DNA strand was clearly modified and therefore not base-paired with adenine in RNA in complexes stalled at position +20 indicates that the RNA-DNA hybrid encompasses not more than 12 bp (Fig. 5). We therefore conclude that the length of the RNA-DNA hybrid is between 8 and 12 bp during early elongation of archaeal transcription.
Comparison of Mechanistic Characteristics of Archaeal, Eukaryotic, and
Bacterial RNAP—Although the basic mechanism of transcription and
general structure of RNAP are highly conserved among the three kingdoms of
life also distinct mechanistic and structural differences exist. The data
described here provide first evidence for the dimensional parameters of a
transcribing archaeal RNAP. As discussed in this paper the archaeal PIC and
the Pyrococcus complex stalled at +5 are likely to extend over the
DNA region from –42 to +18. A very similar DNA section extending from
–55 to +18 is protected in the open complex formed by E. coli
RNAP (17,
34). The upstream part from
–55 to –14, designated as recognition domain, is only partially
protected in the E. coli open complex. After synthesis of 11 bp this
recognition domain is completely dissociated from the DNA whereas the size of
the second DNA domain, the melted domain, remains constant
(34). The recognition domain
of DNA bound by E. coli RNAP seems to be associated with the
transcription factors TBP/TFB in the archaeal system. A major transition at
registers +10/+11 was also observed in both systems. In E. coli, this
transition is characterized by the dissociation of , and the complex
extends at this register from –3 to +27. The archaeal RNAP has also
initiated promoter clearance at this register and extends over 28 bp from
position –4 to +24. Thus, the overall dimensions of the archaeal and
bacterial complex stalled at +11 are very similar. A further contraction of
E. coli RNAP-binding site stalled at register +20 to 22 bp has been
observed (34). By contrast, an
RNAP-binding site of 29 bp was found in archaeal complexes stalled at register
+20. This exo III footprints of archaeal complexes stalled at +20 equal
footprints of pol II stalled between registers +20 and +23 which extend over
31–35 bp (35).
The distance of the catalytic center C to the front edge F of the footprint is constant in active and not retracted bacterial and pol II complexes. The archaeal C-F values determined here were also constant at various registers but with 11–12 nucleotides shorter than in the bacterial (C-F = 18 (25)) and pol II system (C-F = 18–20 (13)).
In all domains of life a characteristic mechanistic similarity is the transition around register +10. At this point all RNAP seems to reach the elongation-committed state. We have not studied abortive products of the archaeal enzyme here but have clearly shown that complexes containing 5–9 nucleotides can be isolated and are fully elongated. This is a common property of the archaeal enzyme and pol II. By contrast E. coli RNAP which, at most promoters, is in an initiation state very similar to the open complex until position +10 and produces reiteratively abortive products in early registers without release and rebinding of RNAP.
From analyses of translocation of the transcription bubble three
characteristic transitions have been postulated in the pol II system
(15,
16). The first transition is
open complex formation. Similar to the archaeal system the eukaryotic open
complex ranges from –9 to +2. By contrast the archaeal RNAP is able to
catalyze DNA strand separation in the absence of TFIIH helicase activity and
ATP (6)
(Fig. 4). We have no evidence
that the second transition in the pol II initiation complex at register +4
characterized by insensitivity of the complex to ATPS
(15) occurs also in the
archaeal system. In the pol II system the region of the initially open complex
readopts the double-stranded conformation between registers +9 to +11, and
this was described as the third transition. Reclosure of the most upstream
part of the archaeal open complex was also observed in these registers. Both
the archaeal RNAP and pol II seem to start promoter clearance around register
+10. In E. coli and pol II complexes continuous opening of the
downstream part of the open region and discontinuous reclosure of the upstream
part have been described (15,
18). The data shown here seem
to indicate that failure to observe continuous reclosure of the upstream end
may not be a mechanistic property of the elongation process but rather due to
the presence of additional complexes stalled at an earlier register which
might mask reclosure at the upstream edge
(Fig. 2A).
The size of the RNA-DNA hybrid is within 9–12 bp in a similar range as in eukaryotic and prokaryotic elongation complexes analyzed by comparable methods (5, 20, 36).
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FOOTNOTES |
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Present address: Universität Regensburg, Lehrstuhl für
Mikrobiologie, Universitätsstr. 31, D-93053 Regensburg, Germany.
To whom correspondence should be addressed. Tel.: 49-941-943-3160; Fax:
49-941-943-2403; E-mail:
Michael.Thomm{at}Biologie.Uni-Regensburg.de.
1 The abbreviations used are: PIC, preinitiation complex; pol II, polymerase
II; NLS, N-lauroyl-sarcosine; RNAP, RNA polymerase; nt, nucleotide;
TBP, TATA box-binding protein; ATPS, adenosine
5'-O-(thiotriphosphate); TFB, transcription factor B.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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