(Received for publication, November 18, 1996, and in revised form, January 22, 1997)
From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037
Stalled Xenopus RNA polymerase I (pol I)
elongation complexes bearing a 52-nucleotide RNA were prepared by
promoter-initiated transcription in the absence of UTP. When such
complexes were isolated and incubated in the presence of
Mg2+, the associated RNA was shortened from the 3-end, and
mono- and dinucleotides were released. Shortened transcripts were still associated with the DNA and were quantitatively reelongated upon addition of NTPs. The cleavage activity could be removed from the pol
I-ternary complex with buffers containing 0.25% Sarkosyl. These
findings indicate that a factor with characteristics similar to
elongation factor TFIIS is associated with the pol I elongation complex. However, addition of recombinant Xenopus TFIIS to
Sarkosyl-washed pol I elongation complexes had no effect, whereas it
showed the expected effects in control reactions with identically
prepared pol II elongation complexes. The results thus suggest the
existence of a pol I-specific cleavage/elongation factor. I also report the sequence of a novel type of Xenopus TFIIS. The
predicted amino acid sequences of the present and previously identified
Xenopus TFIIS are less than 65% conserved. Thus, like
mammalian species, Xenopus has at least two highly
divergent forms of TFIIS.
The process of transcribing a gene can be divided into three main functional steps: initiation, elongation, and termination of transcription. For the eukaryotic ribosomal genes, transcribed by RNA polymerase I (pol I),1 the cis-acting DNA elements comprising the promoter and the terminator have been identified for several species from yeast to man, and trans-acting protein factors required for transcription initiation and termination have been purified and in many cases molecularly cloned (1-5). On the other hand, relatively little is known about the process of transcription elongation by pol I. Due to the considerable length of the ribosomal genes and in light of the finding that at least some pol I even transcribe through the entire intergenic spacer (6), elongation of pol I transcription is likely to be an important process and can be expected to be regulated as well. Indeed, a previous study with Xenopus oocytes provided evidence that there are two types of ribosomal gene transcription, which differ in their elongation characteristics (7). The only pol I elongation factor identified to date is TIF-IC, a mouse pol I transcription factor that is involved in both initiation and elongation (8).
Much more is known about transcription elongation by pol II, and
several protein factors affecting different steps of the elongation
process have been identified. TFIIF and SIII, for example, stimulate
the rate of elongation (9, 10), whereas TFIIS (SII) promotes
read-through of pol II at arrest or pause sites on the template (11).
Others, like TFIIH and TFIIE, are required for promoter escape, an
early step in elongation (12). The present study is concerned with
TFIIS and TFIIS-like factors. cDNAs encoding TFIIS have been
isolated from various species, including Xenopus (Refs. 13
and 14, and this study). TFIIS binds to pol II in the elongation
complex and activates an intrinsic RNase activity that shortens the
nascent transcript from the 3-end (15, 16) (reviewed in Ref. 17). The
transcript cleavage is a hydrolytic reaction and liberates mono- and/or
short oligonucleotides depending on the conformation and location of
the paused elongation complex (18, 19). The shortened transcript can be
reextended in the presence of NTPs indicating that the 3
-ends of the
shortened transcripts are still associated with the catalytic site of
pol II. It is thought that RNA cleavage is important for the observed activation of read-through by TFIIS possibly by allowing the arrested elongation complex to make several attempts to overcome an obstacle. Whether these in vitro observations reflect the in
vivo role of TFIIS is an unresolved question. In yeast, TFIIS is
not essential for viability, but cells lacking TFIIS are sensitive
toward 6-azauracil (20, 21).
TFIIS is generally considered to be specific for pol II. Thus, TFIIS purified from mouse cells did not affect pol I in nonspecific transcription assays (22), and in a different study TFIIS was found to bind to pol II but not to pol III (23). However, another early paper reported that partially purified yeast TFIIS stimulated both pol I and pol II (24). The question of the pol specificity of TFIIS has not been reinvestigated by looking at the more defined effects of TFIIS on paused elongation complexes. In search of pol I-specific elongation factors, I tested whether evidence for an involvement of TFIIS or a TFIIS-like factor in transcription by pol I could be obtained. I used the cleavage of nascent transcripts in a stalled pol I elongation complex as an assay for such a factor. My results show that there is indeed an activity associated with pol I ternary complexes that has similar functional characteristics as TFIIS but is distinct from TFIIS.
Xenopus S-100 cell extract was fractionated over DEAE-Sepharose and heparin-Sepharose as described (25). In the present preparation all the components required for transcription initiation by pol I co-eluted from heparin-Sepharose between 0.4 and 0.8 M KCl (H-0.8 fraction). The protocol described by Dignam et al. (26) was used to prepare a nuclear extract from the Xenopus cell line. To remove endogenous nucleotides, the nuclear extract was precipitated twice with ammonium sulfate (0.33 g/ml of extract) and dialyzed against buffer D (0.1 M KCl, 20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).
Transcription templates were immobilized on Dynabeads (Dynal Corp.) as described (27). For pol I transcription, plasmid pXlr245 (28) was biotinylated at a HindIII site 317 bp downstream of the transcription initiation site, followed by restriction with PvuII 1663 bp upstream of the initiation site in the vector pBR322. To create a heterogeneous population of pol II complexes, plasmid pSP-HSV106 containing a 3.58-kilobase BamHI fragment of herpes simplex virus (29) in vector pSP64 was biotinylated at the HindIII site in the multiple cloning site, and a second cut was made with KpnI, resulting in two biotinylated fragments of 1.39 and 5.19 kilobases.
Immobilized DNA (100-200 ng) was incubated in a 25-µl reaction
containing 10 µl of H-0.8 fraction, 6 mM
MgCl2, 90 mM KCl, 20 mM HEPES, pH
7.9, 10% glycerol, 0.1 mM EDTA, 2 mM
dithiothreitol, and 100 µg/ml -amanitin. After 15-30 min at room
temperature, 50 µM ATP, 50 µM GTP, 0.33 µM [
-32P]CTP (3000 Ci/mmol), and 4 mM phosphocreatine were added to initiate transcription.
(Final concentrations of the components in the reaction are given.)
After 15 min, the reaction was chased with 0.1 mM each of
ATP, GTP, and CTP and 100 µg/ml heparin. pol II transcription
reactions were similar, except that the MgCl2 and KCl
concentrations were 10 and 60 mM, respectively, and no
-amanitin was present. After another 15 min, the template beads were
washed four times in 100 µl of wash buffer as indicated in the figure legends. Wash buffer consisted of 23 mM HEPES, pH 7.9, 90 mM KCl, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 100 µg/ml bovine serum albumin, and 0.05% Nonidet P-40. Magnesium buffer was wash buffer plus 8 mM
MgCl2, EDTA buffer was wash buffer plus 1 mM
EDTA, and Sarkosyl buffer was EDTA buffer plus 0.25% Sarkosyl. In the
reactions in which the isolated complexes were incubated with
recombinant Xenopus TFIIS, two washes in Sarkosyl buffer
were followed by two washes in EDTA buffer. Washed beads were either
directly processed for analysis of the labeled RNA or incubated in a
volume of 25 µl under the conditions indicated in the figure legends.
After treatment with proteinase K/SDS and extraction with
phenol/chloroform, the RNA was concentrated by precipitation with
ethanol and analyzed on 12% (acrylamide:bisacrylamide, 29:1)
polyacrylamide-sequencing gels. For the analysis of the cleavage
products, the beads were washed three times in EDTA buffer and one time
in a buffer containing 50 mM KCl, 10 mM HEPES,
pH 7.9, and 0.5 mM dithiothreitol. They were incubated in
10 µl of the same buffer supplemented with 8 mM
MgCl2, and the reaction was stopped by the addition of 15 µl containing 0.25% SDS, 12 mM EDTA, 250 µg/ml
proteinase K, and 250 µg of carrier RNA. After extraction with
phenol/chloroform and chloroform, the sample was dried in a SpeedVac
concentrator. RNA samples were analyzed on 25% (25:3)
polyacrylamide-sequencing gels.
CpG
(Sigma) was 5-labeled with polynucleotide kinase and
[
-32P]ATP using standard protocols.
[
-32P]CMP was produced from [
-32P]CTP
by boiling in 0.75 N HCl as described in Ref. 30.
To isolate a
cDNA clone encoding the Xenopus homologue of TFIIS, a
degenerate oligonucleotide encoding QTRSADEP
(CAAACTCGT(A/T)(G/C)TGCNGATGA(A/G)CC) and a degenerate oligonucleotide
complementary to the coding sequence for CGNRWKF
(AACTTCCATC(G/T)(A/G)TTNCC(A/G)CA) were used to amplify from
Xenopus genomic DNA a 71-bp fragment using Taq
DNA polymerase (see Fig. 6). The 71-bp product was labeled by
additional rounds of amplification with 32P-5-end-labeled
primers and used to screen a Xenopus liver cDNA library
under high stringency hybridization conditions. A positive clone was
isolated, sequenced, and found to contain an open reading frame of 292 amino acids with high similarity to other reported TFIIS sequences.
For expression of recombinant proteins, a BglII site was
added upstream of the protein-coding sequence by oligonucleotide-primed synthesis using Pfu DNA polymerase (Stratagene). The
fragment from the BglII site to a natural HindIII
site at nucleotide position 268 was ligated to a
HindIII-EcoRI fragment containing the remainder of the coding sequence plus 164-bp downstream flanking sequences and
cloned into the pET28a(+) expression vector (Novagen). The sequence of
the in vitro synthesized section was reconfirmed. Deletion
mutants were created by using naturally occurring restriction sites.
Deletion of a StuI-SspI fragment replaced 59 amino acids from the C terminus with the four residues DLGL (mutant
1), and deletion of a PstI-PstI fragment led
to the internal deletion of residues 45-171 (mutant
2).
Hexahistidine-tagged proteins were expressed and purified as described
(31) except that lysis buffer contained 20 mM imidazole and
no DNase I. Likewise, the nickel-nitrilotriacetate-agarose column was
equilibrated in lysis buffer containing 20 mM imidazole,
and Xenopus TFIIS was eluted with 250 mM
imidazole.
To
generate stalled Xenopus pol I elongation complexes, a
minigene construct containing the ribosomal gene promoter and the first
115 bp of the ribosomal precursor-coding region was immobilized on
magnetic beads by a streptavidin-biotin linkage (Fig. 1) and transcribed in a chromatographic fraction that supported specific transcription initiation by pol I. The first U residue at the 5-end of
the Xenopus 40 S ribosomal precursor is at position 17, followed by a U triplet at positions 53-55. Transcription in the
absence of UTP was therefore expected to yield stalled elongation
complexes carrying transcripts of 16 and, due to unavoidable traces of
UTP in the reaction, 52 nt in length. These transcripts and the
corresponding stalled complexes were termed C16 and G52 according to
the type and position of the last incorporated nucleotide (see Fig. 1).
Analysis of the template-associated RNA showed that the C16 and G52 RNA
were indeed formed (Fig. 2, lane 2; for C16, see
Fig. 3A, lane 7 and Fig.
3B, lane 8). Because a high concentration of
-amanitin was present in all reactions and because in the transcription system used the formation of specific run-off products was dependent on an intact pol I promoter (data not shown, see also
Ref. 27), the C16 and G52 transcripts are interpreted to represent
stalled pol I elongation complexes. Lanes 3 and 4 show that the G52 complex was still elongation-competent. If UTP was added to isolated G52 complexes, they elongated for three nucleotides to position U55 (lane 3). If all four NTPs were added, the
label was quantitatively chased into the 317-nt run-off (lane
4).
Sarkosyl Removes an Endogenous TFIIS-like Activity
In the
reactions analyzed in Fig. 2 (lanes 2-4), the template
beads were washed in Sarkosyl buffer; this process stripped the ternary
complex of most if not all associated proteins. If the beads were
washed in magnesium buffer, the template-associated RNA was found to be
shortened by about 10 nt (Fig. 2, lane 5) suggesting that a
cleavage activity was active during the washing in magnesium buffer
(which takes 5-10 min) and that this activity was removed by the
Sarkosyl buffer. This shortened transcript was still in an active
elongation complex, since it could be reextended to the G52 RNA upon
incubation with ATP, CTP, and GTP (lane 7). The finding that
the G52 complex could be regenerated also demonstrated that the G52 RNA
was shortened at the 3-end. Reextension reactions in the presence of
only two of these three NTPs are shown in lanes 8-10. Based
on these "walking" experiments, the major cleavage product in the
magnesium-washed complexes was found to be the G43 complex. Extension
of the G43 complex with ATP and GTP resulted in the A44 complex
(lane 8), and extension with ATP and CTP yielded the C46
complex (lane 9). Incubation of the G43 complex in the presence of GTP and CTP showed some persisting G43 RNA as expected from
the sequence, whereas other transcripts were shortened back to G35
(lane 10). In addition, some extension to what appears to be
position C51 was observed, probably due to a small amount of
contaminating ATP (lane 10). If all four NTPs were added to the complexes washed in magnesium buffer, all the cleavage products were chased into run-off RNA (lane 11). Prolonged incubation
in magnesium buffer without NTPs led to further degradation of the template-associated transcripts (lane 6). None of these
shortened, and partially reextended transcripts were released from the
template (lanes 13, 14, and 16-19).
Transcript release from the ternary complexes was observed only when
chased to the end of the template (lanes 12 and
15).
To test whether the endogenous cleavage activity requires divalent
cations, complexes were first washed in EDTA buffer. Fig. 3A, lane 2, shows that after washing in EDTA
buffer the G52 RNA was still intact and the result was very similar to
the one obtained with Sarkosyl buffer (lane 7). Incubation
of the EDTA-washed G52 complexes in the magnesium buffer led to rapid
shortening of the associated RNA (lanes 3-5). The time
course identifies the G48/49 and G43 RNA as major metastable
intermediates in the cleavage reaction. Under the present labeling
conditions the shortest G52-derived cleavage product that could be
detected on the autoradiographs was about 20 nt long. Addition of ATP,
GTP, and CTP again led to reextension of the shortened transcripts to
the G52 RNA (lane 6). The finding that transcripts in a pol
I ternary complex can be shortened and reextended in a
magnesium-dependent reaction suggests that the catalytic
site of pol I is involved in the process. Consistent with this notion
is the result that, like pol I transcription elongation, the cleavage
reaction was resistant to high concentrations of -amanitin (200 µg/ml, lane 12). Interestingly, the amount of RNA in the
C16 complex decreased only slightly during incubation in the magnesium
buffer, and no cleavage products were detected. Additional data showed
that elongation of the C16 complex was also less quantitative; whereas
about 70% of the C16 RNA was elongated to U17 RNA upon addition of UTP
(data not shown), this was significantly less than the almost 100% of
the G52 complexes that reelongated (see Fig. 2, lanes 3 and
4). The basis for the apparent difference between the G52
and C16 complex remains to be investigated.
To determine the Sarkosyl concentration required for dissociation of the cleavage activity, stalled pol I complexes were washed with EDTA buffers containing increasing concentrations of Sarkosyl and then incubated in magnesium buffer for 5 min. The result shows that a slight inhibition of the cleavage was observed after washing in 0.1% Sarkosyl (Fig. 3B, lane 6), whereas transcript shortening was fully abolished after washing in a buffer containing 0.25% Sarkosyl (lane 7).
The Transcript-shortening Reaction Liberates Mono- and DinucleotidesTo learn more about the mechanism of the cleavage
reaction, it was important to identify the released products. The
reactions were therefore analyzed on high percentage polyacrylamide
gels, which had previously been used to separate mono- and
dinucleotides (32). My standard transcription protocol employed a chase
with ATP, CTP, and GTP after labeling of the initiated transcripts with
[-32P]CTP at low NTP concentrations. Therefore, the
G52 RNA was expected to be labeled predominantly near its 5
-end.
Because shortening of such RNA from its 3
-end would not yield labeled
cleavage products until late in the reaction, I also performed
transcription reactions without the chase. Fig.
4A, lane 2, shows that these low
nucleotide conditions caused most of the elongation complexes to stall
before C16. Based on the template sequence (see Fig. 1), the two major additional stalled complexes were tentatively identified as C10 and
C14. Incubation in magnesium buffer led to a decrease in the amount of
RNA associated with C10 and C14 and the concomitant appearance of a
cluster of bands that migrated slower than the CTP/CMP markers
(lanes 2-5). Note that again the C16 complex was relatively
unaffected. If "chased" complexes were incubated in magnesium
buffer, these same bands also appeared, but as predicted, later in the
reaction (lanes 6-9). The slowest of these cleavage products co-migrated with the pCpG marker (lanes 14 and
16). Note that pGpC and pCpG cannot be distinguished in this
gel system. The faster bands in the cluster might represent pCpC and
pApC, which are known to migrate ahead of pCpG (32). All of these dinucleotides could be generated from both G52 and C14 RNA. The identification of these cleavage products as 5
-phosphorylated dinucleotides was supported by the finding that all bands disappeared upon digestion of the sample with calf intestinal phosphatase, and a
new cluster of bands with much reduced mobility was generated (lanes 17 and 18). This dramatic shift in
mobility upon removal of the 5
-phosphate from dinucleotide
diphosphates has previously been described (32).
Due to the high amount of input [-32P]CTP, some
residual CTP as well as inorganic phosphate was present in all lanes
even after extensive washing of the beads in EDTA buffer. However, the
time course clearly showed that CMP, which runs slightly behind CTP, was absent at time 0 (Fig. 4B, lane 1) and was
formed during incubation of the cleavage reactions (Fig. 4B,
lanes 2-4). The kinetics of appearance of the CMP band
paralleled the appearance of the dinucleotide bands in both the chased
and the not chased reactions (see Fig. 4A). These
co-migration and phosphatase digestion data thus suggest that
nucleoside 5
-monophosphates and 5
-phosphorylated dinucleotides are
the main products of the cleavage activity associated with stalled pol
I elongation complexes.
The data described in the previous section
indicate that pyrophosphorolytic cleavage of the nascent
transcript does not make a significant contribution to the
observed transcript shortening. Nevertheless, I wondered whether
RNA in stalled pol I complexes would undergo pyrophosphorolysis and how
this reaction would compare with the shortening by the endogenous
Sarkosyl-sensitive activity. Pyrophosphorolysis is known to remove
nucleoside 5-monophosphates from the 3
-end of nascent RNA in ternary
complexes to produce NTPs. Stalled complexes that had been washed in
Sarkosyl buffer were incubated in the presence of 2 mM
sodium pyrophosphate and Mg2+. Fig. 3A,
lanes 7-9, shows that pyrophosphate resulted in rapid shortening and disappearance of the G52 RNA. With longer incubation times, even the C16 RNA underwent pyrophosphorolytic degradation (Fig.
4A, lanes 19-22). Pyrophosphorolytic shortening
went through different metastable intermediates than the endogenous
cleavage reaction (compare lane 8 in Fig. 3A with
lanes 3-5). Furthermore, analysis on a high percentage
polyacrylamide gel showed that dinucleotides (Fig. 4A,
lanes 20-22) and CMP (Fig. 4B, lanes
8-10) were not formed. Since Sarkosyl buffer was more efficient
in removing the input [
-32P]CTP (Fig. 4B,
lane 7), this analysis also revealed that, as expected, CTP
was regenerated in the presence of pyrophosphate (Fig. 4B,
lanes 8-10). Thus, the combined results strongly indicate that the endogenous cleavage reaction is hydrolysis and not
pyrophosphorolysis of the RNA.
The results
described so far suggest that an activity that is very similar to the
pol II elongation factor TFIIS is associated with the pol I elongation
complex. To test whether this activity is identical to TFIIS, I
investigated the effect of recombinant Xenopus TFIIS on
stalled pol I elongation complexes. As a positive control, I prepared a
heterogeneous population of stalled pol II elongation complexes by
transcription of an immobilized plasmid in a Xenopus nuclear
extract in the absence of UTP, followed by washing of the template
beads in Sarkosyl buffer (Fig. 5, lane 1). The
RNA associated with these stalled pol II complexes ranged in size from
about 10 to 60 nt. If all four NTPs were present during transcription,
high molecular weight RNA was formed (lane 2). The synthesis
of both stalled and run-off transcripts was sensitive to 5 µg/ml
-amanitin, indicating that they were the result of transcription by
pol II (lanes 3 and 4). The RNA in the stalled
pol II complexes was not affected by a 1-h incubation in EDTA or
magnesium buffer (lanes 7, 8, and 14). Incubation
in magnesium buffer containing Xenopus TFIIS led to a
shortening of the template-associated transcripts (lanes 10,
15, and 16), whereas the same amount of
Xenopus TFIIS in EDTA buffer had no effect (lane
9). The specificity of the Xenopus TFIIS-induced transcript shortening was demonstrated by testing two deletions of the
recombinant protein. Both a deletion of the C-terminal 59 amino acids
containing the zinc ribbon (33) as well as a large internal deletion
abolished the transcript cleavage activity (lanes 11 and
12). Furthermore, Xenopus TFIIS-induced
transcript cleavage was sensitive to 5 µg/ml
-amanitin (lane
17). These results show that the recombinant Xenopus
TFIIS used in the present study was active and had the expected
characteristics and effects on Sarkosyl-washed, stalled pol II
elongation complexes. If Xenopus TFIIS or one of the mutant
proteins was added to identically prepared pol I elongation complexes,
no effects were seen (lanes 18-21).
TFIIS is a transcription factor that was originally identified as
an activity that stimulates nonspecific transcription by purified pol
II (34). It was later classified as an elongation factor because it
suppresses pausing and increases the yield of long transcripts in
specific transcription reactions (23, 35). TFIIS exerts its function in
elongation by helping stalled or arrested pol II to read through
various transcriptional blockages (11, 36, 37). In stalled and arrested
elongation complexes, TFIIS stimulates the cleavage of the nascent
transcripts at the 3-end and a concomitant backward movement of the
catalytic site of pol II (15, 16). This cleavage reaction is thought to
be important or even required for efficient read-through at these arrest sites.
It is reasonable to postulate that the transcription of ribosomal genes
also requires an elongation factor. As discussed in the Introduction,
the available data appeared insufficient to fully rule out that TFIIS
itself would also serve as an elongation factor for pol I. The
stimulation of the pol-associated transcript cleavage activity is a
more diagnostic assay for TFIIS than the stimulation of nonspecific
transcription. I therefore investigated whether TFIIS would stimulate
the cleavage of nascent transcripts in a stalled pol I elongation
complex. My data show that recombinant Xenopus TFIIS, when
added to Sarkosyl-washed pol I elongation complexes that stalled after
transcription of a 52-nt RNA, did not stimulate cleavage of this
transcript. This negative result was substantiated by control
experiments with Sarkosyl-washed pol II elongation complexes, where the
expected effects of Xenopus TFIIS were readily detected.
Furthermore, Xenopus TFIIS showed all the known
characteristics in these pol II controls, like Mg2+
dependence, -amanitin sensitivity, and dependence on the zinc ribbon
domain near its C terminus.
Whereas the present results make an involvement of TFIIS in pol I
transcription very unlikely, they also show that a different activity
that is similar to TFIIS is present in the pol I elongation complex.
Like TFIIS, this activity shortens the nascent RNA from the 3-end
releasing mono- and dinucleotides. The shortened transcripts are still
associated with the catalytic site of pol I, since they are readily
reextended upon addition of the appropriate NTPs. Finally, the cleavage
activity is removed from the pol I elongation complex with 0.1-0.25%
Sarkosyl; the same conditions also remove TFIIS from the pol II
elongation complex (38). Unlike the TFIIS-induced RNA cleavage reaction
in pol II ternary complexes, the pol I-associated cleavage reaction is
resistant to high concentrations of
-amanitin, a finding that is
consistent with an involvement of the catalytic site of pol I in the
transcript shortening.
In stalled or arrested pol II elongation complexes a low level of RNA cleavage is taking place even in the absence of an auxiliary factor like TFIIS, indicating that the nucleolytic activity resides in pol II (15-17, 39). For pol III this type of hydrolytic cleavage is the only type that has been observed so far, and a TFIIS-like, dissociable activity like the one described in this study has not been identified in pol III complexes (30). In the present experiments I did not detect an intrinsic activity of pol I that would be active without an auxiliary factor. It can be expected, however, that pol I by itself would also show some nucleolytic activity if the sensitivity of the assay was increased. In any case, it is clear that for both pol II and pol I, TFIIS or a TFIIS-like factor is required for efficient transcript cleavage.
After submission of the original version of this manuscript, the
identification of a yeast activity that shortens nascent pol I
transcripts from the 3-end was reported (40). Whereas this yeast
factor and the present Xenopus activity clearly seem to be
related, the available data are insufficient to decide whether they
represent homologous protein factors. It will be interesting to
determine the Mg2+ requirement and the Sarkosyl sensitivity
of the yeast factor as well as the size of the liberated cleavage
products. Apparent differences lie in the chromatographic behavior and
in the finding that the yeast activity can be detected even in the
presence of nucleotides.
Sequences encoding Xenopus TFIIS were recently reported by Kugawa et al. (13) and Plant et al. (14). The latter paper identified two genes for Xenopus TFIIS, termed xTFIIS.oA and xTFIIS.oB, which were interpreted to represent the two gene copies present in the two homologous genomes of the tetraploid Xenopus laevis. The two predicted proteins are 91% identical. The deduced protein sequence reported by Kugawa et al. (13) is 100% identical to xTFIIS.oA. Interestingly, the sequence of the cDNA isolated for the present study, which I will refer to as xTFIIS.l, is very different from both xTFIIS.oA and xTFIIS.oB (Fig. 6). The predicted xTFIIS.l and xTFIIS.oA/B proteins are only 68-70% identical in the 82-amino acid long N-terminal domain and 72-74% identical in the 175-amino acid C-terminal domain. The regions between these two domains are not conserved and of different lengths (46 versus 35 amino acids). The similarity between these two Xenopus proteins is thus about the same as between TFIIS from different vertebrate species. Using reverse transcription-polymerase chain reaction, I found that mRNAs encoding xTFIIS.oA/B and xTFIIS.l are both expressed in a Xenopus tissue culture cell line.2 Xenopus thus has two highly divergent forms of TFIIS, which do not appear to reflect the duplication of its genome during evolution. This finding has a precedent in mammalian species, where in addition to the general form of TFIIS a testis-specific type was identified (41, 42).
It will be interesting to see whether the present TFIIS-like factor belongs to the TFIIS family of proteins. In addition, it will be important to investigate whether and how it changes the properties of the pol I elongation complex. The experiment shown in Fig. 2 shows that in the presence of NTPs a pol I elongation complex is able to elongate to the end of the template regardless of whether it contains this factor (lanes 4 and 11). Furthermore, on the template that was used no intrinsic arrest or pause sites were uncovered during elongation of Sarkosyl-washed complexes. The purification and cloning of this factor will be necessary to investigate its role, if any, in transcription elongation by pol I.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U57837[GenBank].
I thank Ronnie Childs for very valuable technical assistance during the cloning and expression of Xenopus TFIIS.