(Received for publication, September 18, 1996, and in revised form, December 10, 1996)
From the Department of Microbiology and Immunology,
State University of New York, Health Science Center at Brooklyn,
Brooklyn, New York 11203, the
Public Health Research
Institute, New York, New York 10016, and ** The Rockefeller
University, New York, New York 10021
The GreA and GreB proteins of Escherichia
coli induce cleavage of the nascent transcript in ternary
elongation complexes of RNA polymerase. Gre factors are presumed to
have two biologically important and evolutionarily conserved functions:
the suppression of elongation arrest and the enhancement of
transcription fidelity. A three-dimensional structure of GreB was
generated by homology modeling on the basis of the known crystal
structure of GreA. Both factors display similar overall architecture
and surface charge distribution, with characteristic C-terminal
globular and N-terminal coiled-coil domains. One major difference
between the two factors is the "basic patch" on the surface of the
coiled-coil domain, which is much larger in GreB than in GreA. In both
proteins, a site near the basic patch cross-links to the 3 terminus of RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domain
of one protein was fused to the C-terminal domain of the other. In the
hybrid molecules, both the coiled-coil and the globular domains
contribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of
transcript cleavage is determined by the N-terminal domain. These
results implicate the basic patch of the N-terminal coiled-coil domain
as an important functional element responsible for the interactions
with nascent transcript and determining the size of the RNA fragment to
be excised during the course of the cleavage reaction.
Two closely related Escherichia coli proteins, GreA and
GreB, participate in RNA polymerase (RNAP)1
transcription elongation by preventing and/or suppressing the condition
of elongation arrest (1, 2). In addition, both factors have been shown
to facilitate the transition of RNAP from the stage of abortive
initiation to productive elongation (3). GreA and GreB may also have a
proofreading role in transcription (4). It is thought that the Gre
activity is accomplished by endonucleolytic cleavage of RNAs within the
ternary elongation complexes (TECs) (2). The cleavage is followed by
dissociation of the 3-terminal fragment and restart of elongation from
a newly generated 3
-OH terminus (2). Similar reactions are induced in
eukaryotic TECs of RNA polymerase II by transcription elongation factor
TFIIS (5-8), which performs the same functions as GreA and GreB but
lacks any sequence similarity.
GreA and GreB have almost the same molecular mass and share substantial
amino acid sequence homology (2). However, there are several
differences in their functional and biochemical properties in
vitro. First, in all studied TECs that are susceptible for cleavage reactions, GreA induces hydrolysis of short RNAs 2 or 3 nucleotides long from the RNA 3 terminus ("type A" cleavage activity), whereas GreB stimulates hydrolysis of RNAs that are 2-18
nucleotides long, depending on the stage of transcription elongation
("type B" cleavage activity) (2, 9). Second, GreA can only prevent
the formation of arrested TEC (read-through activity), whereas GreB,
besides displaying read-through activity, can convert the preformed
arrested TEC to a productive complex by a cleavage and restart
mechanism (2). Finally, we have recently shown that Gre factors form
reversible complexes with RNAP core or holoenzyme with apparent
Kd values that differ by at least 2 orders of
magnitude: 2-3 × 10
5 M (GreA) and
1-2 × 10
7 M (GreB). These values were
estimated by size exclusion high pressure liquid chromatography of RNAP
complexes with 35S-labeled Gre proteins and were further
supported by affinity chromatography of 35S-labeled Gre
factors on immobilized His-tagged RNAP (10). Similar Kd values were obtained for the complexes of Gre
proteins with purified TECs (prepared on a ribosomal rrnB P1
promoter DNA) carrying 6-mer and 12-mer transcripts (10).
TFIIS and Gre factors do not cleave free RNA or RNA·DNA duplexes (2,
10) and require the formation of a ternary complex between RNAP, DNA,
and RNA transcript for their activity (10) or a specific binary complex
between RNAP and RNA (11). On the other hand, both E. coli
RNAP and eukaryotic RNAP II show a weak ability to cleave RNA in the
absence of factors, and this activity is stimulated by mild alkaline pH
(12) or pyrophosphate (13). These data suggest that the transcript
cleavage factors are not nucleases per se but cofactors that
stimulate an intrinsic nucleolytic activity of RNAP. A working model
that emerged from these data envisages the RNAP catalytic center as the
performer of the cleavage reaction (12, 13). In this model, the
catalytic center disengages from the RNA 3 terminus, slips back along
the transcript, hydrolyzes or pyrophosphorolizes an internal
phosphodiester bond and reengages with the newly generated 3
terminus.
The model is consistent with the notion of conformational flexibility
of the TEC that is central to the discontinuous mechanism of RNA
polymerization (14, 15).
Understanding the mechanism by which Gre proteins induce these perturbations within the TEC and stimulate cleavage would help unravel the mechanism of elongation. In a previous paper, we reported the 2.2-Å resolution crystal structure of GreA (16), which suggested a way this protein might interact with the TEC. The GreA structure consists of the C-terminal globular and N-terminal extended coiled-coil domains, resembling a fist with an outstretched index finger. One surface of the molecule is uniformly negatively charged, while the opposite surface is essentially hydrophobic with a small basic patch near the tip of the finger. We suggested that during interaction with the TEC, GreA's acidic side is oriented away from the acidic surface of RNAP, while the basic patch contacts RNA in the complex.
In the present work, this model was further explored through comparative analysis of GreA and GreB domain organization and function. To this end, we sought to relate the similarities and differences between biochemical functions of the two factors to the common and divergent features of their structures.
Nucleoside triphosphates, RNase-free BSA,
sequencing grade endoproteinases ArgC, AspN, and thrombin were from
Boehringer Mannheim. [-32P]CTP and
[
-32P]GTP (3000 Ci/mmol) were purchased from ICN.
8-N3ATP, 2-nitro-5-thiocyanobenzoic acid (NTCB), and
BNPS-skatole were from Sigma. Prestained protein molecular weight markers were from Amersham Corp.
E. coli
greAgreB
strain AD8571 (12)
was used for overexpression of wild type (WT) and mutant Gre factors
and for preparation of GreA/GreB-free RNAP. E. coli strain
XL1-Blue (Stratagene) was used for selection of all plasmids after
ligation. pTRC99A (Pharmacia Biotech Inc.) was used as an expression
vector for construction of plasmids overproducing WT and mutant Gre
proteins. Plasmid pCF3 is a derivative of pUC19 containing an insertion
of the cloned greB gene (2). All sequences of WT and mutant
gre genes described in this work were verified by dideoxy
chain termination sequencing of the double-stranded plasmid DNA.
The following oligonucleotides were used
for construction of overexpressing plasmids and PCR mutagenesis
(italicized nucleotides correspond to greA and
greB genes, as indicated in parentheses; underlined
nucleotides define the restriction sites of the enzymes, shown in
parentheses; boldface nucleotides correspond to the region coding the
His6 tag): 1) 5-pCAAGCTATTCCGATGACCTTA-3
(GreA 5
terminus); 2)
5
-CGCGC
TTACAGGTATTCCACCTTAATTAC-3
(GreA 3
terminus,
); 3)
5
-GCGCG
AAACGCCCCTGGTTACC-3
(GreB 5
terminus,
); 4)
5
-CGCGC
TTACGGTTTCACGTACTCGATAGC-3
(GreB 3
terminus,
); 5)
5
-CGCGC
TTAGTGATGGTGATGGTGATGCAGGTATTCCACCTTAATTAC-3
(GreA 3
terminus,
); 6)
5
-CGCGC
TTAGTGATGGTGATGGTGATGCGGTTTCACGTACTCGATAGC-3
(GreB 3
-terminus,
).
-Oligonucleotides 1 and 2, synthesized
according to the DNA sequence of the greA gene (17), were
used for PCR amplification of greA from E. coli
DNA. The PCR product was purified by electrophoresis and digested with
BamHI. The expression vector pTRC99A was digested with
NcoI, filled in by Klenow fragment of DNA polymerase I, and then cleaved by BamHI. The greA fragment was
inserted into the expression vector pTRC99A between the blunt end and
BamHI sites. Ligation of these fragments yielded plasmid
pMO1.1, which contained the greA gene under the control of
isopropyl-1-thio--D-galactopyranoside-inducible trc promoter. For engineering of the plasmid overproducing
GreB, oligonucleotides 3 and 4 were used for PCR amplification of the greB gene from the plasmid pCF3. The purified PCR product
was digested with BspHI and BamHI and inserted
into linearized pTRC99A between the NcoI and
BamHI sites. Ligation of these fragments yielded pMO1.4,
which contained the greB gene under the control of the
trc promoter. Plasmids overproducing His6-tagged
GreA (pMO1.1His) and GreB (pMO1.4His) were obtained similarly, except
that oligonucleotides 5 and 6 carrying His6 codons at the
end of each gre gene were used for PCR.
We engineered a set of 10 chimeric GreA/GreB molecules that carry N-terminal polypeptide
fragments of one Gre protein fused to the C-terminal part of the other
at amino acid positions 6, 12, 31, 81, and 107 of GreA. Ten hybrid
primers were synthesized: the 5-proximal half of each primer
corresponded to the DNA fragment of one gre gene immediately
upstream of the fusion and the 3
-proximal half corresponded to the
fragment of the other gre gene immediately downstream of the
fusion. All greA/greB hybrids were obtained by a
conventional two-step PCR procedure.
-WT GreA and GreB were purified to
apparent homogeneity as described (18). For isolation of
His6-tagged GreA,
greAgreB
E. coli (AD8571) cells transformed with pMO1.1His were grown and
induced with isopropyl-1-thio-
-D-galactopyranoside as
described (18). The cells were pelleted by low speed centrifugation,
homogenized in 5 ml of buffer A (7 M guanidine HCl, 40 mM Tris-HCl, pH 7.5, 0.8 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol) and
centrifuged at 25,000 × g for 20 min. The supernatant
was added to 0.8 ml of Ni2+-chelating NTA-agarose beads
(Qiagen) preequilibrated in buffer A. The suspension was incubated for
20 min at 25 °C on a rotary shaker, and the beads were pelleted by
low speed centrifugation and washed with 3 × 10 ml of buffer A. The adsorbed GreA was renatured by five subsequent washings of the
beads with 5 ml of buffer B (the same as buffer A but without guanidine
HCl) and eluted with 2 ml of buffer B, containing 0.6 M
imidazole. The eluate was desalted and concentrated in Centricon-10
(Amicon). The resulting electrophoretically homogeneous
His6-tagged GreA (1.2 mg in 200 µl) was stored at
20 °C in buffer B containing 50% glycerol.
His6-tagged GreB and GreA/GreB hybrids were purified
essentially as described above.
GreA/GreB-free RNAP holoenzyme (,
) was isolated from
E. coli AD8571 strain, and RNAP carrying an insertion of 130 amino acids in
-subunit (
insRNAP) was isolated from
E. coli MKDC747 strain as described (12).
The read-through, antiarrest,
and transcript cleavage assays were performed using a 202-base pair
E. coli rrnB P1 DNA fragment as described previously (1, 2,
18, 30) (see "Results" and legends to Figs. 3 and 7 for
details). The NTP concentrations for chain extension reactions were 5 µM except for 8-N3ATP, which was 100 µM. The RNA products were analyzed, autoradiographed, and
quantified by PhosphorImager (Molecular Dynamics) as described (12, 18).
Gre-RNAP Binding Assay
15 µg of each
His6-tagged Gre factor were incubated for 10 min at 4 °C
with 7 µl of Ni2+-chelating NTA-agarose beads in 20 µl
of buffer C (40 mM Tris-HCl, pH 7.5, 30 mM KCl,
0.1 mM EDTA, and 0.1 mM dithiothreitol)
containing 0.8 M NaCl. The beads were then washed in 3 × 500 µl of the same buffer. The yield of immobilized Gre factors was
~95% for all proteins. 5 µg of RNAP holoenzyme in 20 µl of
buffer C containing 0.1 M NaCl, 10 mM
MgCl2, and 0.5 mg/ml of BSA were added to the beads and
incubated with gentle shaking for 15 min. Free RNAP was removed by
centrifugation, and the beads were washed twice with 100 µl of the
same buffer. The beads carrying adsorbed RNAP were suspended in 20 µl
of transcription buffer (buffer C, supplemented with 0.5 mg/ml BSA and
10 mM MgCl2) containing 0.5 µg of
rrnB P1 DNA template, 0.5 mM CpA, 2 µM ATP, and 1 µM
[-32P]CTP. After incubation at 37 °C for 10 min,
the beads were washed with 4 × 200 µl of buffer C containing
0.2 mg/ml of BSA and 0.2 M NaCl. The adsorbed proteins and
TEC carrying radiolabeled hexameric transcript
CpApCpCpApC (6C-TEC) (here and elsewhere, boldface type symbolizes radioactive phosphates) were eluted
from the beads with 50 µl of buffer C containing 20 mM EDTA and 0.8 M NaCl. An aliquot of each eluate (5 µl for
all samples except 0.5 µl for WT GreB) was analyzed by denaturing
23% polyacrylamide gel electrophoresis (PAGE) followed by
autoradiography and quantitation by PhosphorImager. In the control
experiment, 6C-TEC was prepared as above but in the absence of beads,
and His6-tagged GreA or GreB (100 µg/ml) were mixed with
2 µg of RNAP in 20 µl of transcription buffer. The resulting 6C-TEC
was purified by gel filtration using Quick-Spin G-50, and an aliquot
(1/200 of the total material) from each reaction was analyzed by
PAGE.
Photocross-linking
experiments were performed using radioactively labeled 9A*-TEC carrying
8-N3AMP at the RNA 3 terminus essentially as described
previously for the cross-linking of GreA (16).
The
cross-linked -subunit (~3 × 104 cpm) carrying
radioactive RNA was purified by SDS-4% PAGE as described (19) and
mixed with 5 µg of purified unlabeled
-protein. The resulting
material was precipitated by cold acetone (1:1 (v/v)), redissolved in 4 µl of buffer D (40 mM Tris-HCl, pH 8.2, 40 mM
NaCl, 1 mM dithiothreitol, 1 mM EDTA)
containing 1% SDS, and incubated for 5 min at 37 °C. The mixture
was diluted with 200 µl of buffer D containing 0.15 µg of thrombin
and incubated at 37 °C for 5 h. The reaction was terminated by
the addition of 10 µl of 10 mM diisopropylfluorophosphate and analyzed by Tris-glycine SDS-14% PAGE followed by autoradiography. For proteolytic cleavage of GreB, the reaction mixture containing cross-linked GreB (1 × 105 cpm) was mixed with 4 µg
of unlabeled GreB and incubated with 0.1 µg of AspN protease in 50 µl of buffer D containing 5 mM CaCl2 for
1 h at 30 °C. Under these conditions, the cross-linked
was
resistant to proteolysis by AspN (data not shown). The reaction was
terminated by the addition of EDTA (20 mM final
concentration) and analyzed by Tris-Tricine SDS-14% PAGE and
autoradiography. The 16-kDa proteolytic fragment carrying cross-linked
RNA was purified as described above for
and digested with 1 µg
of endoproteinase ArgC in buffer D containing 10 mM
CaCl2 and 10 mM dithiothreitol for 4 h at
37 °C. The reaction was terminated and analyzed as described above
for AspN.
The purified cross-linked species were mixed with 5 µg of the same purified unlabeled protein prior to chemical degradation. For cleavage at Cys, the proteins were treated with NTCB for 4 h at 37 °C as described (20). For cleavage at Trp, the proteins were incubated with BNPS-skatole (10 mg/ml) in 70% acetic acid for 2 h, at 37 °C according to Ref. 21.
CD SpectrometryCD spectra were obtained using an AVIV circular dichroism DS-62 spectrophotometer at 4 °C. The buffers were 50 mM sodium phosphate, pH 7.5, containing 50 mM NaCl for GreA and 250 mM NaCl for GreB. Protein concentrations used for experiments were in the range of 15-40 µM. The path length of the cuvette was 0.1 cm. Data were collected every 1 nm with a bandwidth of 1.5 nm, and the CD signal was averaged over 2 s. Each spectrum was averaged over four scans from 190 to 300 nm. The temperature dependence of the CD profiles was studied at 222 nm using a cuvette with the path length of 0.1 cm, and the temperature was varied from 5 to 60 °C in 2 °C increments. The samples were equilibrated for 2 min at each temperature point, and the CD signal was averaged over 30 s.
Protein concentration was determined using Bradford's protein assay (22). Tris-glycine and Tris-Tricine SDS-PAGE was performed according to Laemmli (23) and Schagger et al. (24), respectively, and gels were stained with Coomassie Brilliant Blue R-250.
Diffraction quality crystals of GreB have not been obtained yet. However, the high degree of functional (2) and sequence homology (35% sequence identity) between E. coli GreA and GreB suggests that they have similar structures (25). Moreover, examination of the sequence alignment reveals that key features of the GreA structure are conserved in GreB (16). These features include hydrophobic residues that participate in forming the hydrophobic core of the coiled-coil domain; charged residues that form interhelical salt bridges that stabilize the coiled-coil structure; all of the residues involved in interactions between the N- and C-terminal domains; and the helical bulge at position 24, causing a skip in the coiled-coil heptad repeat pattern.
Ultraviolet CD spectroscopy provides a rough indicator of the secondary
structure of globular proteins. The measured CD spectra of GreA and
GreB are nearly identical (Fig. 1A). The
minima at 208 and 222 nm are indicative of helical secondary structure
(26), as expected from the GreA structure. The
[]222/[
]208 ratio has previously been
used to assess the number of helical strands within a molecule (27). A
value for [
]222/[
]208 of about 0.8 is
associated with single-stranded
-helix, whereas a value of about 1.0 or more is suggestive of a two-stranded coiled-coil. The value of [
]222/[
]208 for GreA and GreB (1.3 for both) is suggestive of double-stranded coiled-coils, as observed in
the GreA x-ray crystal structure. In addition, the CD signals at 222 nm
were monitored as a function of temperature to investigate the thermal
unfolding of the proteins (Fig. 1B). Both proteins behave
similarly, with cooperative thermal unfolding transitions at about 46 and 43 °C for GreA and GreB, respectively.
Taken together, these observations strongly support the conclusion that the structure of GreB is very similar to that of GreA. A three-dimensional model of GreB was calculated using comparative protein modeling by satisfaction of spatial restraints as implemented in the program MODELLER2 (28). First, the relatively high sequence similarity between GreA and GreB resulted in reliable alignment with only one single residue deletion. Second, the alignment was used to derive many distance and dihedral angle restraints on the GreB sequence. Finally, the GreB model was calculated by minimizing the violations of these restraints. The main chain atoms of the model are generally within 0.4 Å of the equivalent GreA atoms. Assuming that the relative orientation of the coiled-coil and the globular domain is conserved, the main chain root mean square error in the model is expected to be approximately 1.5 Å, and about 70% of the side chain rotamers are expected to be modeled correctly (29).
The most striking result from the homology model of the GreB structure
is revealed by examining the charge distribution around the
water-accessible surface (Fig. 2). As noted earlier
(16), the charge distribution around the surface of GreA exhibits a remarkable asymmetry. One face of the molecule is strongly acidic (Fig.
2, top left), whereas the opposite face is neutral except for a small basic region formed by Arg52 and
Arg37. The asymmetry in the GreB charge distribution is
even more dramatic. As with GreA, one face of GreB is acidic (Fig. 2,
bottom left); however, the opposite face is strongly basic
and has a much larger basic patch than GreA. These structural features
may be related to the different functional properties of the two
factors.
Cross-linking of RNA 3
To investigate the orientation of GreA and GreB relative to
RNA in the TECs we employed RNA-protein cross-linking using a photoreactive analog of AMP (8-N3-AMP) incorporated into
the 3 terminus of radioactively labeled nascent RNA. On the ribosomal rrnB P1 promoter (starting sequence: CACCACUGACACGG ...
) in the presence of the dinucleotide CpA, ATP, and
[
-32P]CTP, RNA polymerase forms a stable TEC carrying
the hexameric transcript CpApCpCpApC
(6C) (30). The 6C-TEC was purified by gel filtration (Fig.
3A, lane 1) and used for stepwise
extension of the transcript to 7U, 8G, and 9A (lanes 2,
3, and 4, respectively). The addition of a
mixture of UTP, GTP, and 8-N3-ATP to radiolabeled 6C-TEC
led to the formation of 9A*-TEC carrying transcript
CpApCpCpApCpUpGpA* (lane 5)
with photoactive azido probe at the RNA 3
end.
In the absence of Gre proteins, irradiation of the 9A*-TEC generated a
single radioactive band visible on an autoradiograph of SDS-PAGE (Fig.
3B, lane 6), which corresponds to the
cross-linked -subunit of RNAP. A control experiment was performed
with a mutant RNAP carrying a 127-residue insertion in a nonessential region of the
-polypeptide (
ins) (31). The
ins-subunit migrates in SDS-PAGE much slower than the WT
(32) (Fig. 3C, lanes 1 and 2).
However, cross-linking experiments conducted with both WT and
insRNAP produced a single radioactive band of the same mobility (lanes 5 and 8, respectively),
eliminating
as the possible cross-linked species. The conclusion
that the 3
end of RNA cross-links to
is in agreement with our
earlier result obtained for the 22A*-TEC formed on T7AI promoter
(19).
The addition of GreB to the 9A*-TEC followed by UV-irradiation resulted
in a 3-fold decrease of cross-linking and the appearance of a
second radioactive band, which migrates slightly above GreB with an
apparent molecular mass of 19 kDa (Fig. 3B, lane
4). The yield of GreB cross-linking was approximately 18% of the
initial 9A*-TEC. Under the same conditions, GreA cross-linked with the yield of about 5% and did not cause decrease in the cross-linking of
(Fig. 3B, lane 5). Fig. 3C
demonstrates that neither GreA (lanes 4 and 7)
nor GreB (lanes 3 and 6) induced the
cross-linking of the
-subunit. The cross-linked sites on GreB and
were mapped by specific chemical and enzymatic degradation as
shown in Figs. 4 and 5.
Mapping of the RNA-cross-linking Site in GreB
First, the cross-linked GreB was subjected to limited proteolysis with endoproteinase AspN, which specifically hydrolyzes peptide bonds at the N-terminal side of Asp (33). Under nondenaturing conditions, this enzyme quantitatively cleaves free GreB only at Asp47 generating two polypeptide fragments, Met1-Ala46 and Asp47-Pro158, as revealed by N-terminal amino acid sequencing (data not shown). The reaction yielded a radioactive product with an apparent molecular mass of 16 kDa (Fig. 4A, lane 1), which is the expected mass of the C-terminal fragment Asp47-Pro158 plus the RNA probe. Next, the 16-kDa fragment was chemically cleaved at Cys residues by NTCB (20). Since GreB contains only one Cys at position 68, the only expected cleavage products are the 2.5-kDa N-terminal and 10.5-kDa C-terminal fragments. Two radioactive bands were detected by autoradiography after SDS-PAGE analysis: the uncleaved GreB and a peptide with an apparent molecular mass of ~5.0 kDa (Fig. 4B, lane 2), which we interpreted as the N-terminal fragment carrying an additional mass of 2.5 kDa contributed by the cross-linked 9A-RNA. These results map the cross-link within the fragment Asp47-Lys67. This conclusion was confirmed by a chemical cleavage of the 16-kDa proteolytic fragment at Trp residues with BNPS-skatole (21). This cleavage resulted in a single radioactive product with an apparent mass of ~6.5 kDa (Fig. 4C, lane 2), which could only correspond to Asp47-Trp91. For further mapping, the 16-kDa fragment was digested by endoproteinase ArgC, which specifically cleaves peptide bonds at the C-proximal side of Arg (34). The major radioactive product observed near the 3.5-kDa marker (Fig. 4C, lane 3) corresponds to Asp47-Arg63 or shorter fragments within. These results localize the site of the photocross-link to the 17-amino acid-long Arg-rich segment between Asp47 and Arg63, which coincides with the location of the large basic patch of GreB.
In our previous paper (16), we described the localization of the
cross-linked site on GreA in 9A* TEC between Asp41 and
Phe57. Thus, the cross-linking of the RNA 3 terminus to
both Gre factors occurs within an overlapping 22-amino acid-long
fragment between positions 41 and 63, suggesting that this region is
responsible for the interactions with the nascent RNA 3
terminus.
For localization of the cross-link in , the initial
radioactive material was isolated from SDS gel and subjected to
exhaustive degradation by thrombin. This enzyme recognizes the
consensus sequence Leu-Val-Pro-Arg-Gly-Ser. However, it is also highly
specific for the sequence Arg-Gly and cleaves peptide bonds at the
C-proximal side of Arg (35). After a prolonged incubation in the
presence of SDS, the reaction yielded a single prominent radioactive
band visible on an autoradiogram after SDS-PAGE (Fig. 5A,
lanes 1-4) with apparent molecular mass of ~20 kDa.
Further analysis of the isolated 20-kDa fragment by Tris-Tricine
SDS-PAGE revealed two closely migrating bands with masses of ~20 and
~19 kDa (Fig. 5B, lane 1). Taking into account
the additional mass of RNA these could only correspond to fragments
Gly733-Arg907 and
Gly746-Arg907. Next, the mixture of the two
Gly-Arg fragments was subjected to chemical cleavage at Trp residues
by BNPS-skatole. The reaction yielded two radioactive cleavage
products: a band corresponding to the detached RNA and a band with a
mass of ~6 kDa (Fig. 5B, lane 2), which we
interpreted as the C-terminal fragment
Trp869-Arg907. Finally, the initial
cross-linked
was chemically cleaved at Cys residues by NTCB. The
major radioactive product with a mass of ~10 kDa, representing about
70% of the total radioactivity (Fig. 5C, lane 2)
was observed, which was also the smallest fragment visible on the
autoradiograph. According to the distribution of Cys residues in
(see Fig. 5D), the 10-kDa product corresponds to a fragment
flanked by Cys814 and Cys898. The other
radioactive bands above the 10-kDa product visible on the
autoradiograph presumably correspond to the products of incomplete
digestion, since the yield of cleavage reactions with NTCB may vary
from 90 to 30% (20), depending on the susceptibility of Cys residues.
However, the possibility of minor cross-linking sites (representing
less than 10% of the total radioactivity in cross-linked
) outside
of the localized Cys814-Cys898 fragment cannot
be excluded. Thus, the major cross-link site is mapped to the 29-amino
acid-long segment between Trp869 and Cys898.
This segment is located near the conserved "region G" of
, where we have previously mapped the cross-link site for the RNA 3
terminus in the 22A*-TEC formed on a T7A1 DNA (19).
Since the azido probe is directly attached to the adenine at the RNA 3
end and has a reactivity radius of less than 5 Å, the simultaneous
cross-linking of
and Gre proteins strongly suggests that they are
situated in close proximity to each other.
To delineate functional domains in Gre proteins, we constructed a set of chimeric GreA/GreB molecules carrying the N-terminal fragment of one Gre protein fused to the C-terminal fragment of the other. Each GreA/GreB hybrid carried six His residues at the C terminus in order to facilitate their purification. His-tagged GreA, GreB, and GreA/GreB hybrids (purified as described under "Experimental Procedures") were compared in their ability to activate the transcript cleavage reaction in TECs formed on a ribosomal E. coli rrnB P1 DNA fragment (2, 18).
The transcript cleavage assays used in these experiments are based on
two properties of Gre proteins: first, the "catalytic" nature of
their activity and, second, the type specificity of their respective
activities. Gre proteins are able to induce complete transcript
cleavage in TECs in substoichiometric amounts even when they are
present at a Gre/TEC molar ratio of 0.01:1 (1). This catalytic property
of Gre factors enables us to quantitate their specific activity in
enzymological terms. Thus, in quantitative assays, one unit of cleavage
activity is defined as the amount of Gre protein required for the
hydrolysis of 50% of the RNA in 7U- or 9A-TECs under standard reaction
conditions (18). The specific cleavage activity is expressed as
units/µg of Gre protein. In 9A-TEC carrying the radiolabeled
transcript CpApCpCpApCpUpGpA, GreA stimulates cleavage and
release of dinucleotides pGpA and pCpU (type A cleavage
activity), whereas GreB induces the cleavage of the tetranucleotide
pCpUpGpA (type B cleavage activity) (2). Thus, in the
qualitative assay performed on the 9A-TEC, the type of products
generated define the GreA- or GreB-type activity. The results obtained
from comparative analyses of hybrid and WT Gre proteins using these
criteria are summarized in Fig. 6.
For hybrids 1 and 6, in which only the first six N-terminal residues were exchanged, the type of cleavage activity was determined by the C-terminal part of the resulting polypeptide. Thus, hybrid 1 retained the GreA-type activity, while hybrid 6 retained the GreB-type activity. Both hybrids exhibited decreased cleavage activity toward 7U- and 9A-TECs compared with the corresponding WT Gre factors. The extent of the decrease varied from 7- to 1000-fold, depending on the combination of N- and C-terminal fragments and the type of TECs. Hybrid 1 and GreA displayed, for example, 140 and 1000 units/µg cleavage activity toward 7U-TEC, or 0.1 and 100 units/µg toward 9A-TEC, respectively. Hybrid 6 and GreB displayed 20 and 300 units/µg activity toward 7U-TEC or 50 and 1000 units/µg toward 9A-TEC, respectively. Thus, the changes within the short N-terminal segment of GreA and GreB affected the specific activity (units/µg) of these proteins and their selectivity toward TECs but did not affect the type of cleavage reaction.
Increasing the N-terminal portion of GreB in GreA to 12 residues (hybrid 2) restored the original cleavage activity of GreA. However, similar substitution in GreB (hybrid 7) led to a total loss of cleavage activity toward both TECs. Apparently, this effect was due to incorrect protein folding, since hybrid 7, when expressed in E. coli, was found in the inclusion bodies, less than 1% of which was recovered in soluble form after renaturation from 7 M guanidine HCl. The same results were obtained when the first 12 N-terminal residues in GreA and GreB were deleted (data not shown). According to the three-dimensional structure of GreA and GreB, four nonconserved residues at positions 4, 5, 8, and 9 and conserved Thr6 are buried inside the Gre molecule and, presumably, participate in forming the hydrophobic core of the N-terminal coiled-coil domain (16). Our results together with crystallographic data suggest that the N-terminal 12 residues form an essential structural element for initiating the proper folding of the coiled-coil domain of Gre molecules.
The exchange between GreA and GreB of the N-terminal 31 residues
(hybrids 3 and 8) as well as 44 residues (data not shown) yielded
insoluble proteins that refolded poorly and displayed no detectable
cleavage activity. The N-terminal residues 1-44 comprise the first
-helix of the coiled-coil domain, and most of the residues in this
region that are critical for the formation of correct coiled-coil
structure are highly conserved in both Gre molecules (2, 16). The only
exceptions are those that comprise two of the four interhelical bonds
that stabilize the overall structure of N-terminal domain: salt bridge
Lys22-Glu66 and hydrogen bond
Arg25-Asn54 in GreA and salt bridge
Glu25-Arg54 and hydrophobic bond
Trp22-Val62 in GreB (2, 16). Presumably, the
disruption of these two interhelical bonds in GreA/GreB hybrids led to
destabilization of coiled-coil structure and resulted in proteins that
failed to assume their native conformation.
Exchanging the N-terminal 81 residues of GreA with the corresponding residues of GreB (hybrid 4) resulted in the switch of cleavage activity from type A to type B (Fig. 6, hybrid 4; Fig. 7A, lanes 5 and 6). The specific transcript cleavage activity of hybrid 4 toward 7U- and 9A-TECs decreased 1500- and 500-fold, respectively, in comparison with GreB. (Because the cleavage activity of hybrid 4 switched to type B, its specific activity can only be compared with GreB.) Similarly, the hybrid 9 displayed type A cleavage activity (Fig. 6, hybrid 9; Fig. 7A, lanes 7 and 8); however, the specific activity of this hybrid toward 7U- and 9A-TECs decreased only moderately (50- and 3-fold, respectively) compared with GreA. Hybrids 5 (Fig. 7A, lanes 9 and 10) and 10 (Fig. 7A, lanes 11 and 12) displayed the same types of cleavage activity as hybrids 4 and 9, respectively. However, the specific cleavage activity of hybrid 5 showed substantial improvement over that of hybrid 4, whereas the activity of hybrid 10 toward 9A-TEC was lower than that of hybrid 9 (Fig. 6). These data demonstrate that the N-terminal coiled-coil domain of Gre protein dictates the type of cleavage activity characteristic for each factor. These results also suggest that the C-terminal globular domain does not affect the type of cleavage reaction but is required for full transcript cleavage activity toward TECs.
The Antiarrest Activity of Gre Factors Is Determined by Their N-terminal Coiled-coil DomainThe hybrids 4, 5, 9, and 10 were further compared with the WT factors for their ability to suppress the formation of arrested TECs during transcription elongation on a ribosomal rrnB P1 promoter (read-through assay) and to induce the transcript cleavage in preformed arrested TECs (antiarrest assay) (2, 18).
For the read-through assay (Fig. 7B), the radiolabeled 6C-TEC (Fig. 7B, lane 1) was incubated with four NTPs alone or in the presence of GreA, GreB, or hybrid proteins. In the absence of Gre factors, about 50% of the TEC was arrested at positions +12 and +13 (lane 2). The addition of GreA, GreB, or hybrids 5 and 9 to the initial 6C-TEC, prior to the addition of NTPs, reduced the formation of arrested TECs and increased the total amount of the full-length run-off transcription product (lanes 3, 4, 7, and 8, respectively). The hybrids 4 and 10 had little or no effect on the formation of arrested TECs (lanes 5 and 6, respectively).
For the antiarrest assay (Fig. 7C), the arrested 12C- and 13G-TECs obtained by chasing the initial 6C-TEC with four NTPs were further purified by gel filtration (lane 2), and then were exposed to either hybrid or WT factors. Of all the proteins tested, only GreB and hybrid 5 induced the cleavage of arrested TECs (lanes 4 and 9, respectively), yielding TECs carrying 5A and 6C transcripts that could be converted into full-length run-off products upon the addition of NTPs (data not shown). Under the same conditions, GreA and hybrids 9, 10, and 4 did not cleave any arrested TECs (lanes 5, 6, 7, and 8, respectively). The fact that GreA was inactive toward arrested 12C- and 13G-TECs is consistent with our earlier observation that, in contrast to GreB, GreA can act only before the arrest took place and not after the fact (2).
Thus, the functional activity of GreA/GreB hybrids in the read-through and the antiarrest assays was determined by their N-terminal domain and correlated with their specific transcript cleavage activity on 9A-TEC (see Figs. 6 and 7A). Indeed, hybrid 9 carrying the coiled-coil domain of GreA and globular domain of GreB acted as GreA displaying only the read-through activity, and hybrid 5 composed of the coiled-coil domain of GreB and two-thirds of the globular domain of GreA behaved as GreB, exhibiting both the read-through and the antiarrest activities. At the same time, hybrids 4 and 10 were inactive in both assays apparently due to their low specific transcript cleavage activity (see Fig. 6).
Both the Coiled-coil and the Globular Domains Contribute to Specific Binding of Gre Factors to RNAPThe functional roles of Gre domains were further explored by comparing the binding affinities of GreA/GreB hybrids and WT Gre proteins with RNAP. The addition of six His residues to the C terminus of Gre proteins does not alter the binding properties of GreB but causes a ~10-fold decrease in the binding affinity of GreA (10). When Gre factors are immobilized on Ni2+-NTA-agarose through His tags, they are still able to bind RNAP from solution in accordance with their binding affinities. Moreover, RNAP molecules bound to immobilized Gre factors are able to form a stable 6C-TEC on rrnB P1 promoter, and neither GreA nor GreB affects the formation and the stability of 6C-TEC.3 Therefore, the amount of 6C transcript generated by RNAP on the beads is proportional to the amount of RNAP molecules adsorbed to immobilized Gre proteins.
To assess the contribution of each Gre domain to the binding of Gre
molecules to RNAP, Ni2+-NTA-agarose beads carrying WT or
hybrid factors were incubated with RNAP holoenzyme at a molar ratio of
10:1 in the presence of BSA as a carrier protein. After removal of free
RNAP, the immobilized Gre·RNAP complex was incubated with
rrnB P1 DNA, CpA, ATP, and [-32P]CTP to
allow the formation of 6C-TEC. The free NTPs were removed, and the
radioactivity in 6C-TEC formed on the beads was quantified following
urea-PAGE analysis (Fig. 8). According to this
semiquantitative assay, the radioactivity of 6C transcript associated
with immobilized GreB was ~300 times higher than that of GreA
(lanes 7 and 4, respectively). In solution, the
amount of 6C-TEC formed was the same in the presence of GreA and GreB
(Fig. 8, lanes 1 and 2, respectively), indicating that the low recovery of 6C transcript from immobilized GreA
(lane 4) was due to a low amount of RNAP bound to GreA.
These results are in good agreement with our earlier observation that
the apparent Kd values for GreB·RNAP and
GreA·RNAP complexes differ by approximately 2 orders of magnitude
(10). When Ni2+-NTA-agarose was used for the assay without
immobilized Gre factors, no radioactive 6C transcript was detected
(lane 3). In comparison with GreA, hybrids 4, 5, 9, and 10 displayed a 15-, 50-, 8-, and 4-fold increase in the apparent binding
affinity toward RNAP (Fig. 8, lanes 5, 6,
8, and 9, respectively), although none of them reached the binding efficiency of GreB. Both hybrids 5 and 9, carrying,
respectively, the N- and C-terminal domains of GreB, have higher
binding affinity than GreA, suggesting that the sites responsible for
specific binding to RNAP are equally distributed between the two
domains of the Gre molecule. These results also suggest that the
binding of Gre factors to RNAP requires cooperative action of their
coiled-coil and globular domains.
The transcript cleavage reaction appears to be a ubiquitous and evolutionarily conserved function among multisubunit RNA polymerases. It has been observed in TECs formed by vaccinia virus RNA polymerase (36), prokaryotic RNAP of E. coli (2), and eukaryotic RNA polymerases II (5-8) and III (37). The elongation factor TFIIS facilitates the transcript cleavage and the read-through/antiarrest activities of RNA polymerase II in such divergent organisms as yeast (8), insects (6), and mammals (5). In prokaryotes, the genes encoding Gre factors have been identified and sequenced from five different organisms including E. coli (2, 17), Hemophilus influenzae (38), Rickettsia prowazekii (39), Mycobacterium leprae,4 and Mycoplasma genitalium (40). In addition, GreA- and GreB-like activities were detected in Pseudomonas sp., Acinetobacter sp., and Bacillus subtilis.3 The biological importance of Gre factors is also underscored by the fact that the greA gene is present in the smallest known genome of any free-living organism, M. genitalium (40).
The amino acid sequence alignment of seven homologous GreA and GreB
proteins is shown in Fig. 9. Although the percentage of identical residues among the members of the Gre family varies, most of
the key structural elements of the E. coli GreA (16) are
preserved with few exceptions. The conserved structural elements include hydrophobic residues at the expected a and
d positions of the characteristic heptad repeat,
(abcdefg)2, and at positions 6 and 72 that form
the hydrophobic core of the N-terminal domain (Fig. 9, yellow
shading) and most of the residues participating in interhelical
and interdomain salt bridges and/or hydrogen bonds stabilizing the
overall Gre structure (magenta and orchid
shading). In addition, all five members of the GreA family contain
two highly conserved basic residues at positions 37 and 52 (turquoise shading), comprising a small basic patch on the
water-accessible surface of GreA (see Fig. 2, top).
Similarly, two representatives of the GreB family contain six conserved
basic residues at positions 2, 52, 53, 56, 60, and 67 (Fig. 9,
blue shading) that form a large basic patch on the surface
of GreB (see Fig. 2, bottom). These observations suggest
that all Gre proteins have similar structural organization and spatial
arrangement of their N- and C-terminal domains. Comparison of the CD
spectra of E. coli GreA and GreB and homology modeling of
the GreB three-dimensional structure strongly support this
conclusion.
Our results of RNA-protein cross-linking demonstrate that both GreA and
GreB interact with the 3 end of RNA and presumably
-subunit of
RNAP in TEC through a 16-17-residue-long peptide segment that overlaps
with basic patches (Fig. 9, red underline). This supports
our earlier hypothesis that the surface of Gre molecules harboring the
basic patch contacts RNAP and RNA in TEC (16). The analysis of the
functional roles of Gre domains using Gre hybrids revealed that the
N-terminal domain is responsible for specific binding of Gre factors to
RNAP and carries the structural determinants conferring the GreA or
GreB type of cleavage activity and, presumably, the antiarrest and
read-through activities. The C-terminal domain does not have a direct
role in transcript cleavage or antiarrest function but participates in
binding to RNAP and is required for full activity of Gre factors.
This work was initiated while D. K., M. O., and S. B. were at A. Goldfarb's lab at the Public Health Research Institute. We thank A. Goldfarb and V. Nikiforov for generous support and advice and J. Lee for critical reading of the manuscript. We are also grateful to W. McAllister, A. Das, and R. Landick for comments and D. Cowburn and S. Cahill for help with CD measurements.