(Received for publication, July 10, 1995; and in revised form, September 25, 1995)
From the
RNA polymerase II arrested at specific template locations can be rescued by elongation factor SII via RNA cleavage. The size of the products removed from the 3`-end of the RNA varies. The release of single nucleotides, dinucleotides, and larger oligonucleotides has been detected by different workers. Dinucleotides tend to originate from SII-independent complexes and 7-14 base products from SII-dependent complexes (Izban, M. G., and Luse, D. S.(1993) J. Biol. Chem. 268, 12874-12885). Different modes of cleavage have also been recognized for bacterial transcription complexes and are thought to represent important structural differences between functionally distinct transcription intermediates.
Using an elongation complex ``walking'' technique, we have observed factor-independent complexes as they approach and become arrested at an arrest site. Dinucleotides or 7-9-base (large) oligonucleotides were released from SII-independent or dependent complexes, respectively. The abrupt shift between the release of dinucleotide versus large products accompanied the change from factor-dependent to factor-independent elongation, as described by others. However, not all factor-independent complexes showed cleavage in dinucleotide intervals since oligonucleotides 2-6 bases long were also liberated from elongation-competent complexes. These were all 5`-coterminal oligonucleotides indicating that a preferred phosphodiester bond is targeted for cleavage in a series of related complexes. This is consistent with recent models postulating a large product binding site that can hold RNA chains whose size increases as a function of chain polymerization. A specific transitional complex was identified that acquired the ability to cleave in a large increment one base insertion event prior to attaining the arrested configuration.
Elongation by RNA polymerase II (pol II) ()is a
complex process that is poorly understood. Accessory elongation factors
have been identified that influence the elongation reaction in
vitro (Kane, 1994). Elongation factor SII allows transcription to
proceed in vitro from template locations that are intrinsic
arrest sites. At such sites RNA polymerase II remains potentially
active but unable to elongate RNA chains even though a full complement
of nucleotide substrates is present. SII interacts with arrested
complexes and activates a nascent RNA cleavage activity that is thought
to reside in RNA polymerase II itself (reviewed in Reines(1994)). RNAs
are shortened from their 3`-end; the 5`-fragment remains in an
elongation complex and can be re-extended. RNA cleavage is required for
the resumption of chain elongation from specific template sites (Reines et al., 1992; Izban and Luse, 1993b). The requirement for SII
can apparently be circumvented in vitro by
pyrophosphate-mediated RNA cleavage (Rudd et al., 1994). The
probability of arrest upon chain re-extension is not altered by chain
cleavage. Instead, repeated rounds of RNA shortening and re-extension
provide additional opportunities for RNA polymerase II to bypass an
arrest site, most or all of which are only partial blockages to
elongation (Gu et al., 1993; Izban and Luse, 1993b; Guo and
Price, 1993).
RNA polymerase from Escherichia coli also has an elongation factor-activated nuclease involved in readthrough of transcriptional blockages (Surratt et al., 1991; Borukhov et al., 1992, 1993; Nudler et al., 1994; Lee et al., 1994; Feng et al., 1994). Two elongation factors that associate with E. coli RNA polymerase, GreA and GreB, activate distinct forms of the cleavage reaction. GreA induces cleavage of 2- and 3-base oligonucleotides while GreB activates releases of large oligonucleotides (Borukhov et al., 1993). This reaction appears to fulfill a similar role in chain elongation to that activated by SII.
A full understanding of the RNA polymerase II-associated ribonuclease requires a characterization of the reaction products. One report indicates that mononucleotides are released from isolated elongation complexes arrested within the adenovirus major late transcription unit (Wang and Hawley, 1993). Other workers report that di- and oligonucleotides are the major products of cleavage (Izban and Luse, 1993a, 1993b; Guo and Price, 1993). It is clear, however, that the cleavage increment is variable. It is a function of both the extent to which elongation is SII-dependent and the sequence of the DNA template (Izban and Luse, 1993a, 1993b). For example, extension of a template strand A tract at an arrest site increases the fraction of elongation complexes that become arrested. RNA cleavage releases proportionally more large oligonucleotide than dinucleotide during transcription on such templates (Izban and Luse, 1993b). The variable size of the cleavage product is probably related to the physicochemical differences between arrested and elongation-competent complexes.
Here we have investigated the characteristics of the cleavage reaction carried out by a nested set of elongation complexes on a portion of a human histone gene. We confirm the preference of SII-independent complexes for removing dinucleotides. However, 5`-coterminal oligonucleotides from 3 to 9 bases long were also released from distinct complexes by scission of a preferred phosphodiester bond. Arrested complexes preferentially removed 7-9-base oligonucleotides by cleavage at this position, proving that cleavage of only a single phosphodiester bond was sufficient to rescue these arrested complexes. A hexanucleotide was released from a transitional factor-independent complex located at a long pause site 1-nucleotide incorporation event before an arrest site. Hence, elongation competence of a pol II complex does not always predict the cleavage increment, and acquisition of the ability to cleave in the large increment can precede the arrested phenotype. This transition may presage the change in elongation potential that the complex experiences upon arrest.
Fast protein liquid chromatography-purified
nucleoside triphosphates (NTPs) were purchased from Pharmacia-LKB
Biotechnology (Uppsala, Sweden). Formalin-fixed Staphylococcus
aureus (immunoprecipitin) was obtained from Life Technologies Inc.
Dinucleotides ApC and CpG were purchased from Sigma. Oligonucleotides
CpGpUpUpUpUpU and CpGpUpUpUpUpUpUpU were synthesized by National
Biosciences, Inc. (Plymouth, MN). [-
P]CTP
was obtained from Amersham Corp.
The template used in these reactions contains the core adenovirus major later promoter and a segment of human histone H3.3 gene containing an intrinsic arrest site called Ia. Transcription in the absence of SII results in approximately half of the RNA polymerase II molecules becoming arrested at positions 205, 206, and 207 (Ia) (+1 is the transcription start site (Gu et al., 1993) (Fig. 1, lane 1)). These arrested elongation complexes require elongation factor SII for further RNA synthesis. Detailed studies have revealed that SII activates a nuclease activity that cleaves the nascent RNA near its 3`-end, which is essential for the resumption of polymerization (Reines et al., 1992; Gu et al., 1993). Preliminary mapping experiments showed that no more than 7-9 nucleotides were removed before the relief of arrest and renewed chain elongation (Gu et al., 1993).
Figure 1:
Identification of cleavage products
from arrested (Ia) complexes. Washed Ia complexes labeled uniquely at
position C received: no addition (lane 2); SII
(0.5 µg), MgCl
(7 mM) and
-amanitin (1
µg/ml) (ama, lane 3); SII and MgCl
(lane
4); SII only (lane 5); MgCl
only (lane
6); or SII, MgCl
, and 0.25% Sarkosyl (sark, lane
7) for 10 min at 28 °C. Small cleavage products were separated
from elongation complexes by centrifugation and resolved on a 25% gel (lower panel). RNA remaining in complexes was isolated and
resolved on a 5% gel (upper panel). Lane 1 is
5`-end-labeled RNA showing runoff RNA (RO) and Ia-RNA (Ia). An RNA marker of 260 nucleotides is indicated in lane M. Lane M` shows
P-labeled,
chemically synthesized pCpGpUpUpUpUpU.
To identify the
cleavage products, we have taken advantage of SII-activated nascent RNA
cleavage to label elongation complexes (Gu and Reines, 1995). Arrested
Ia complexes were assembled on linear templates in the presence of
unlabeled NTPs and immunoprecipitated with an anti-RNA antibody (Eilat et al., 1982; Reines, 1991). They were washed free of NTPs,
and SII was added to activate nascent RNA cleavage. From previous work,
we know that Ia-RNA is shortened to yield a major 5`-product ending in
an A residue at position 198 (Gu et al., 1993) (see Fig. 6). In the presence of [-
P]CTP
and GTP, A
complexes could be extended to G
with a single
P atom in the transcript between bases
198 and 199. The G
complexes could be further extended
with UTP to obtain an elongation complex bearing an internally labeled
Ia-RNA. Under these conditions, only Ia-RNA was labeled (Gu and Reines,
1995) (Fig. 1, lane 2). These uniquely labeled
complexes were the substrates for the analysis of SII-dependent
cleavage.
Figure 6:
Summary of the data generated in this
study. The DNA sequence surrounding site Ia is shown at the top. Elongation complexes are shown on the left, and
major labeled cleavage products are shown on the right. Minor
products are shown in parentheses. The product assignments for
complexes U, U
, and U
are
inferred from the collection of products generated by a mixture of
these three complexes. The asterisk indicates the position of
the
P label. Release of pGpA from A
complexes is indicated from prior mapping experiments (Gu et
al., 1993)
After nucleotide depletion and treatment with SII,
cleavage products were separated from the elongation complex by
precipitation with fixed S. aureus. Precipitated RNA was
isolated and resolved on a 5% polyacrylamide gel (Fig. 1, upper panel). Products released from complexes were resolved
on 25% polyacrylamide gels (Fig. 1, lower panel). In
the presence of both Mg and SII, radioactivity was
lost from Ia-RNA and found in 6-9-base oligonucleotides (Fig. 1, lane 4). The formation of these
oligonucleotides was strongly dependent upon SII (Fig. 1, lane 6) and inhibited by
-amanitin (Fig. 1, lane 3) and Sarkosyl (Fig. 1, lane 7). The
lack of complete dependence upon added SII probably resulted from
residual factor used to activate transcript cleavage before labeling.
Two of these small products comigrated with authentic, chemically
synthesized pCpGpUpUpUpUpU (Fig. 1, lanes 4 and M`) and pCpGpUpUpUpUpUpUpU (see Fig. 5A, lanes 7 and M). Therefore, it appeared that
3`-end-labeled Ia complex gave rise to products pCpGpUpUpUpU,
pCpGpUpUpUpUpU, pCpGpUpUpUpUpUpU, and pCpGpUpUpUpUpUpUpU, consistent
with the heterogeneous ends of Ia-RNA (Gu et al., 1993).
Chemically synthesized oligonucleotide pCpGpUpUpUpUpUpUpU was not
degraded when included in a reaction (data not shown) indicating that
the 6-, 7-, and 8-base oligonucleotides we observed were not generated
by secondary digestion of the nonamer. The same products were liberated
by SII-mediated cleavage using rat liver SII (Fig. 2, lanes
1 and 2) or recombinant human SII (Fig. 2, lane 3), both of which were purified to near homogeneity (Fig. 2, right panel and Conaway et
al.(1995)). The loss of radioactivity from these 6-9-base
oligonucleotides after alkaline phosphatase treatment (Fig. 3, lane 7 versus 8) demonstrated that they were 5`-coterminal
resulting from cleavage on the 5`-side of the phosphodiester bond
between bases A
and C
.
Figure 5:
Identification of cleavage products from a
nested set of elongation complexes. A, labeled G complexes were extended with 1 µM (lane 2),
2 µM (lane 3), 4 µM (lane
4), 16 µM (lane 5), 40 µM (lane 6), 400 µM (lane 7) UTP, or
400 µM each of all four nucleotides (lane 8) at
28 °C for 15 min. After washing, these complexes were treated with
rat liver SII (phosphocellulose fraction, 0.5 µg) and 7 mM MgCl
for 10 min. Cleavage products were isolated and
resolved on a 25% polyacrylamide gel. Shown in lane M is
[
P]pCpGpUpUpUpUpUpUpU. Label comigrating with
CTP and CMP is indicated. The amounts of dinucleotide (B);
tri-, tetra-, and pentanucleotide (C); or hexa-, hepta-,
octa-, and nonanucleotide (D) were quantitated as a percent of
total oligonucleotides released in reactions such as those shown in A and plotted as a function of UTP concentration. Note the
expanded y axis of panel
C.
Figure 2:
Cleavage products from Ia complexes
treated with native rat and recombinant human SII. Left, washed Ia complexes labeled at C were treated with
rat liver (RL) SII (phosphocellulose fraction, 0.5 µg, lane 1; Bio-Gel SP-5 PW fraction, approximately 1 ng, lane
2) or recombinant human (rhu) SII (Bio-Gel SP-5 PW
fraction, approximately 6 ng, lane 3). Cleavage products were
isolated and resolved on a 25% polyacrylamide gel. Lane M shows
P-labeled, chemically synthesized
pCpGpUpUpUpUpU. Right, human SII was expressed in E.
coli, purified to near homogeneity, resolved on a SDS gel, and
silver-stained. Two protein standards (Bio-Rad prestained
SDS-polyacrylamide gel electrophoresis standards, low range) are
indicated in kilodaltons.
Figure 3:
Alkaline phosphatase sensitivity of
cleavage products. Complexes C (lanes 3 and 4), G
(lanes 5 and 6), and Ia (lanes 7 and 8) were radiolabeled at position
C
and treated with rat liver SII (phosphocellulose
fraction, 0.5 µg) and 7 mM MgCl
for 10 min at
28 °C. Cleavage products were separated from the complexes by
centrifugation, butanol-extracted, dried, dissolved in 9 µl of 1
mM MgCl
, and treated with (lanes 4, 6, and 8) or without (lanes 3, 5,
and 7) 0.05 unit of calf intestinal alkaline phosphatase
(Boehringer Mannheim) for 10 min at 37 °C. Samples were dried and
resolved on a 25% polyacrylamide gel. Lanes 1, 2, and M are chemically synthesized,
P-labeled pApC,
pCpG, and pCpGpUpUpUpUpU, respectively.
We have
previously employed pol II ``walking'' experiments to
assemble a nested set of elongation complexes on this template
extending from position 199 to 207 (Gu and Reines, 1995). Labeled
C and G
complexes were prepared by
incubating unlabeled A
complexes with
[
-
P]CTP (not shown) or
[
-
P]CTP and GTP (Fig. 4, lane 3, and Fig. 6), respectively. Elongation by G
complexes in the presence of increasing amounts of UTP resulted
in a series of complexes between U
and U
(Fig. 4, lanes 4-7, and Fig. 6).
Each set of complexes was washed free of NTPs and incubated with SII
and MgCl
. The major product released from G
complexes comigrated with authentic pCpG (Fig. 5A, lane 1) and was sensitive to
alkaline phosphatase (Fig. 3, lane 5 versus 6).
Incubation of G
complexes with 1 µM UTP
resulted in a mixture of complexes U
, U
,
and U
(Fig. 4, lane 6). The labeled
3`-products observed from these complexes were predominantly pCpG (72%)
with some trimer (pCpGpU) (10%), tetramer (pCpGpUpU) (9%), pentamer
(pCpGpUpUpU) (5%), and hexamer (pCpGpUpUpUpUpU) (5%) (Fig. 5A, lane 2). Trimer, tetramer, and
pentamer contained
P at their 5` terminus since they were
sensitive to phosphatase treatment (data not shown). Although these
products arose from a mixture of complexes, we infer that the trimer,
tetramer, and pentamer came from the U
, U
,
and U
complex, respectively. In any case, they result
from the cleavage of the phosphodiester bond between A
and C
. The size of the released oligonucleotides
increased as G
complexes were extended toward site Ia
with increasing amounts of UTP (Fig. 5A, lanes
3-6, 5C, and 5D). In particular, the ratio of
hexamer to each of the other oligonucleotides increased commensurate
with chain extension (Fig. 5D). When G
was incubated with 400 µM UTP (and no CTP),
6-9-base oligonucleotides were observed almost exclusively (Fig. 5A, lane 7). Labeled material migrating
faster than pCpG was also observed. A portion of this is pApC resulting
from unextended C
complexes (see below and Fig. 3, lanes 1 and 2). Label co-migrating with CTP and CMP
was identified as well. Both were present in preparations of complexes
prior to SII treatment. CMP in particular could not be removed even
after extensive washing and gel filtration (data not shown) as
described by others (Rudd et al., 1994; Izban et al.,
1995). From this we conclude that CMP can represent at most 10.1
± 0.6% (mean ± S.D., n = 6) of the total
cleavage products identified in the reactions shown in Fig. 5A.
Figure 4:
Assembly and ``walking'' of
elongation complexes. 5`-End-labeled elongation complexes (lane
1) were treated with rat liver SII (phosphocellulose fraction, 0.5
µg) and 7 mM MgCl for 1.5 min at 28 °C to
generate A
complexes (asterisk, lane
2). Radiolabeled G
complexes (lane 3) were
obtained as described under ``Materials and Methods'' and
extended with the indicated amounts of UTP for 15 min at 28 °C (lanes 4-7). RO, runoff
RNA.
When this template is transcribed by pol
II, about half of the elongation complex becomes arrested nearly
equivalently at U, U
, and U
while the other half passes through site Ia (Gu et al.,
1993). Under conditions where RNA pol II was able to proceed past site
Ia, i.e. in the presence of all four NTPs, we observed the
expected release of an approximately equivalent amount of 7-, 8-, and
9-base oligonucleotides (Fig. 5A, lane 8).
Equivalent amounts of hexanucleotide are also present, probably due to
a long pause at site 204 (data not shown). Interestingly, elongation
from this complex, although slow, is SII-independent (Gu et
al., 1993; Gu and Reines, 1995). Hence, this SII-independent
elongation complex released oligonucleotides in a cleavage increment
characteristic of arrested elongation complexes 205-207,
suggesting that this represents a critical transition between
factor-independent and -dependent elongation. In other words, cleavage
in the large increment became uncoupled from the acquisition of the
arrested state.
The effect of withholding CTP (the nucleotide
downstream of site Ia) is to allow the 50% of complexes that would
otherwise read through site Ia to become arrested at position 207.
Therefore, all the complexes become arrested at positions 205, 206, or
207 when G complexes are supplied with only UTP (Gu and
Reines, 1995). Indeed, more 9-base oligonucleotide was generated when
CTP was absent because those complexes that were SII-independent
accumulated at position 207 (Fig. 5A, lane 7 versus
8, and Fig. 6). Hence, there is a good correlation between
arrest at position 207 and generation of the 9-base oligonucleotide by
SII-dependent cleavage.
These results suggested a preferred cleavage
site at the phosphodiester bond between bases 198 and 199 since a
nested set of 5`-coterminal oligonucleotides were released as the chain
was extended from 200 to 207. We next asked what cleavage products were
generated from C complexes that contain an RNA chain 1
base longer than this apparent preferred cleavage boundary.
Surprisingly, a dinucleotide comigrating with authentic pApC was
released after SII treatment (Fig. 3, lane 1 versus 3).
Consistent with this assignment, the electrophoretic mobility of the
product shifted dramatically after alkaline phosphatase treatment, but
its label was not lost since the
P was located at an
internal phosphodiester bond (Fig. 3, lane 3 versus 4,
and Fig. 6). This finding emphasizes the extent to which
dinucleotide release is favored in SII-independent complexes. The
results of this analysis are summarized in Fig. 6.
We have identified the products of nascent RNA cleavage from a well characterized arrested elongation complex. The strategy utilized in this study also enabled us to determine how the site of cleavage varies across a set of related elongation complexes. In our experiments, one nucleotide was labeled in RNA chains ranging from 199 to 207 nucleotides long. This approach allowed us to unequivocally identify labeled cleavage products and in particular to define the sequence of the large oligonucleotides.
SII activates RNA cleavage in both SII-independent and SII-dependent complexes although cleavage is more readily accomplished by arrested complexes (Reines et al., 1992). The product of SII-activated RNA cleavage has been identified by others in different transcription systems. Wang and Hawley(1993) reported that exclusively mononucleotides were released by human pol II elongation complexes. They proposed that this activity may serve as a proofreading exonuclease for RNA synthesis in analogy to some DNA polymerases. Luse and co-workers (Izban and Luse, 1993b) identified dinucleotides and oligonucleotides released by this reaction and found that the size of the oligonucleotides varied as a function of the elongation factor dependence of the complexes. A dinucleotide was the predominant product for SII-independent complexes while 7-14-base oligonucleotides were released from SII-dependent complexes (Izban and Luse, 1993a, 1993b). In that work the sequence identity of the large oligonucleotides was not determined. Guo and Price(1993) also showed that SII stimulated the release of dinucleotides from Drosophila elongation complexes.
Our
findings confirm the work of Luse and co-workers (Izban and Luse,
1993a, 1993b) in showing that elongation complexes arrested at site Ia
released 7-9-base oligonucleotides and elongation-competent
complexes such as C and G
cleaved
predominantly in a dinucleotide increment. The 7-9-base
oligonucleotides were generated from U
, U
,
and U
complexes by cutting the same phosphodiester bond
between bases A
and C
, suggesting that this
is a favored cleavage site for arrested complexes. In addition,
complexes extending from position 199 to 204 yielded 2-, 3-, 4-, 5-,
and 6-base oligonucleotides, all of which have the same 5`-end. Hence,
oligonucleotides of many lengths can be released, although
dinucleotides are the preferred cleavage product by factor-independent
complexes.
Complex U is unusual in that it is largely
SII-independent, yet cleavage removes a 6-base oligonucleotide almost
exclusively. This may represent a transitional complex in that it
carries out cleavage as arrested complexes bearing RNAs 1, 2, or 3
bases longer (205, 206, 207) but is not itself an arrested complex.
Hence, the increment of cleavage cannot necessarily be predicted for a
given elongation complex.
Based on a report that mononucleotides are
removed from some pol II complexes (Wang and Hawley, 1993) and that the
phosphodiester bond between 198 and 199 was a preferred cleavage
position (Fig. 6), we expected that CMP might be released from
C complexes. Surprisingly, a significant amount of the
dinucleotide pApC was observed. Our effort to identify CMP as a product
was hampered by a background of [
P]CMP tightly
associated with labeled complexes, similar to the finding of others
(Izban et al., 1995). Hence, we cannot rule out the
possibility that CMP is a cleavage product, albeit a minor one.
Our
data are consistent with current models developed for the discontinuous
(inchworming) movement of the E. coli elongation complex (Wang et al., 1995; Nudler et al., 1995) and may be related
to why RNA pol II becomes arrested at site Ia. At some DNA sequences
enzyme translocation becomes uncoupled from chain elongation leading to
the development of internal strain in the complex. For a strained E. coli RNA polymerase, transcription terminates when the RNA
structure permits (Wang et al., 1995; Nudler et al.,
1995). T tracts on the nontemplate strand appear to be a common signal
for the inchworming cycle (Nudler et al., 1995) and are found
at many pol II arrest sites (Hawley and Roeder, 1985; Sato et
al., 1986; Kerppola and Kane, 1987; Reines et al., 1987).
In our template, the sequence around arrest site Ia is TTTTTTTCCCTTTTTT
on the nontemplate strand, which is associated with a bend in this
region of the template (Kerppola and Kane, 1990). Previous exonuclease
III footprint analysis showed little or no translocation of pol II
between the A and U
complexes (Gu et
al., 1993). The T tracts could be involved in uncoupling enzyme
translocation and chain extension (an inchworming signal). The unusual
template structure could contribute to the accumulation of strain until
a threshold is encountered whereupon pol II becomes arrested. Strain
accumulation may be gradual but appears to rise most dramatically at
the U
to U
step. As for bacterial RNA
polymerase in the strained configuration, the RNA 3`-end is close to
the downstream edge of the complex (Gu et al., 1993). Similar
uncoupling has also been seen for protein-blocked elongation complexes
(Pavco and Steege, 1990; Kuhn et al., 1990; Reines and Mote,
1993; Nudler et al., 1995). The distance between the 3`-end of
Ia-RNA and the downstream boundary of pol II is unusually short
(10-12 base pairs) compared to that found in elongation-competent
complexes (18-20 base pairs) (Gu et al., 1993). The
inchworming model describes a product groove on the enzyme in which RNA
is polymerized and accessible to elongation factor-activated cleavage
(loose binding site (Chamberlin, 1994; Nudler et al., 1995)).
Elongation of RNA by our complexes from position 198 to 207 appears to
be filling such a groove. We have not detected the upper limit of RNA
that can be accommodated in the groove but it is at least 9 bases. The
capacity of such a product groove could vary between arrest sites. More
experiments will be required to demonstrate if this additional
plasticity exists in elongation complexes.