(Received for publication, December 14, 1995; and in revised form, February 14, 1996)
From the
Factor 2 was previously identified in Drosophila K cell nuclear extract (K
N) as an activity
suppressing the appearance of long transcripts (Price, D. H., Sluder,
A. E., and Greenleaf, A. L.(1987) J. Biol. Chem. 262,
3244-3255). A 154-kDa protein with factor 2 activity was purified
to apparent homogeneity from K
N. An immobilized template
assay indicated that factor 2 caused the release of transcripts by RNA
polymerase II in an ATP-dependent manner. Some early elongation
complexes were resistant to factor 2 action but became sensitive after
treatment with 1 M KCl. In the absence of factor 2,
transcription complexes still exhibited a low degree of processivity
suggesting that factor 2 was only partially responsible for abortive
elongation.
The study of eucaryotic gene expression is aided by the
application of procaryotic paradigms. A major feature of procaryotic
transcriptional control is the default employment of negative
elongation potential, which stops RNA polymerase before a gene is fully
transcribed. Control of expression is accomplished through the action
of positive factors, which alleviate pausing or termination so that
full-length mRNAs are synthesized. Attenuation of the tryptophan operon
in Escherichia coli and anti-termination by bacteriophage
N and Q proteins are two examples in procaryotes in which
expression of operons is regulated by specific blocks to transcription
elongation(1) . Although there are intrinsic signals for
pausing and termination, specific termination factors also play an
important role in the control process. E. coli Rho protein is
the most extensively studied termination
factor(2, 3) . It binds the nascent RNA, uses ATP to
drive translocation along the transcript toward the elongation complex,
and then causes the dissociation of the RNA from the ternary complex.
The interplay of negative and positive factors provides the means to
control transcription elongation.
As is found in procaryotes the control of transcription elongation is also critical for gene expression in eucaryotes(4, 5, 6) . Factor-dependent termination has been observed for eucaryotic RNA polymerases. A specific sequence in the nascent transcript is not sufficient to terminate vaccinia RNA polymerase but requires a viral termination factor identical with vaccinia mRNA capping enzyme(7) . In the transcription systems directed by RNA polymerase I (8, 9) or RNA polymerase III (10) or mitochondrial polymerase(11, 12) , specific termination factors have also been described. No RNA polymerase II termination factor has been identified.
We proposed a
model for the control of elongation by Drosophila RNA
polymerase II that incorporated the function of both negative and
positive factors(13, 14) . The model states that all
RNA polymerase II molecules that initiate from a promoter are destined
to produce only short transcripts due to the action of negative
transcription elongation factors (N-TEF). ()Escape from this
abortive elongation into productive elongation requires the action of
positive transcription elongation factors (P-TEF). Fractionation
studies have recently identified three components required to
efficiently generate productive elongation complexes(15) . One
of these components, P-TEFb, was purified to apparent homogeneity and
was shown to act after initiation(15) . N-TEF was proposed to
function early during transcription and result in pausing and premature
termination(14) . Here we report the purification of factor 2,
a component of N-TEF, from Drosophila K
cells.
To test the effect of factor 2 on washed early elongation complexes during transcription (see Fig. 4), a chase solution was added to allow further elongation in the presence or absence of factor 2. The chase solution brought the reaction to either 60 or 250 mM HMK and 600 µM GTP, ATP, CTP, and UTP. The reactions were stopped with HKE. The labeled transcripts in soluble and bead fractions were isolated and analyzed in 6% denaturing gels(16) .
Figure 4: Function of factor 2 during elongation. Early elongation complexes were generated, washed with either 60 mM KCl (A) or 1 M KCl (B) containing buffers and chased as described under ``Experimental Procedures'' and diagrammed. The transcripts were analyzed with 6% polyacrylamide gel electrophoresis. washed complexes, washed early elongation complexes before the chase. Factor 2 (F2), 0.3 µl (A) or 0.1 and 0.3 µl (B) of glycerol gradient fraction 13 (see Fig. 2), was added as indicated during the chase. B, bound; S, supernatant; nt, nucleotide.
Figure 2: Purification of factor 2. A and D, silver-stained 6-15% gradient SDS protein gel analysis of the indicated fractions generated during the last two steps of purification. B and E, transcriptional analysis for suppression activity as in Fig. 1A except that a template with a 520-nucleotide (nt) runoff was used. C and F, immobilized template assay for transcript release activity as in Fig. 1B except that complexes were washed with 1 M KCl, and 600 µM ATP was used. Only the released transcript fraction was analyzed. -, no addition of factor 2; OP, onput fraction; M, 10-kDa ladder protein marker.
Figure 1: Negative transcription activity of partially purified factor 2. A, titration of factor 2 during in vitro transcription using a continuous labeling protocol detailed under ``Experimental Procedures.'' Increasing volumes of partially purified factor 2 (0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µl) were added. -, no addition; +, addition of 1 µl of partially purified factor 2. Transcripts were analyzed by denaturing 6% polyacrylamide gel electrophoresis. The position of a 1,000-nucleotide runoff is indicated. B, immobilized template assay for transcript release activity. Isolated early elongation complexes were generated by pulse labeling followed by 250 mM KCl wash as described under ``Experimental Procedures'' and diagrammed above the gel. 150 µM ATP was used in the transcript release assay. Released transcripts in the supernatant (S) and bound transcripts associated with the immobilized template (B) were separated and analyzed by denaturing 18% polyacrylamide gel electrophoresis. nt, nucleotides.
Elongation control involves the action of both positive and negative factors(13, 14) . Recently we showed that partially purified Drosophila factor 2 was involved in this process(15) . To further define the role of factor 2 we undertook its purification. We used a set of partially purified factors that reconstructs accurate initiation from a promoter-containing template (15, 16) but does not support the production of DRB-sensitive transcripts because of the lack of P-TEFb(15) . Using this system factor 2 suppressed the appearance of long transcripts(16) .
Since factor 2 suppressed the generation of long transcripts, increased pausing or termination was a possible explanation. An immobilized DNA template was used to differentiate paused from terminated transcripts. Early elongation complexes were isolated and then incubated with partially purified factor 2 in the presence or absence of ATP. Released RNAs were separated from the template-associated transcripts by magnetic concentration. Factor 2 or ATP by itself was not sufficient to release the transcripts associated with early elongation complexes (Fig. 1B). However, in the presence of ATP, partially purified factor 2 caused the release of transcripts associated with the immobilized complexes (Fig. 1B).
Figure 3: Association of factor 2 with elongation complexes. A, early elongation complexes were generated and washed with either low (60 mM KCl) or high salt (1 M KCl) as described under ``Experimental Procedures'' and diagrammed above the gel. 600 µM ATP was used in the termination assay. Either the total or the released transcripts were analyzed as indicated on an 18% polyacrylamide gel. Total transcripts were analyzed after washing and represent the starting material for the termination reactions. nt, nucleotides. B, interaction of factor 2 with high salt washed early elongation complexes. High salt (1 M KCl) washed early elongation complexes were generated as described in A and incubated with purified factor 2 for 2 min at 25 °C. After the incubation, the complexes were washed with low or high salt and analyzed as described in A.
Factor 2 promoted transcript release from high salt washed complexes, but it was not clear if it associated with these complexes. To address this question, high salt washed early elongation complexes were generated and incubated with factor 2 without ATP. After washing again with low salt, these complexes were able to release 60% of the associated transcripts when only ATP was added. This indicated that factor 2 stably associated with the elongation complexes under low salt conditions. Supplementation with additional factor 2 caused a slight increase in the percentage of transcripts released (Fig. 3B). This additional release could have been due to a subsaturating level of factor 2 being used in the first incubation. After washing again with 1 M KCl, most of the transcript release activity was removed, indicating that the association of factor 2 with elongation complexes was not stable to high salt (Fig. 3B). Supplementation of factor 2 restored the transcript release activity. These association studies indicated that the properties of factor 2 were similar to those of the transcript release activity found in the initial low salt washed complexes (Fig. 3A). The simplest explanation is that factor 2 is responsible for all transcript release we observed. Once antibodies to factor 2 are available, it will be possible to determine if factor 2 is present in the initial elongation complexes.
As expected, high salt washed early elongation complexes did not release transcripts during subsequent elongation in 60 mM KCl. Surprisingly, these complexes still encountered blocks to elongation (Fig. 4B). These blocks were substantially relieved by chasing with 250 mM KCl. Evidently, some component of N-TEF seen in low salt washed resistant complexes was retained after the high salt wash. This component of N-TEF was apparently suppressed but not removed by treatment with high salt. When the high salt washed complexes were supplemented with increasing amounts of factor 2, there was a decrease in the length of the transcripts synthesized and an increase in the amount of released transcripts (Fig. 4B). As was seen with the stalled complexes, almost all of the complexes became substrates for factor 2 after high salt treatment.
We purified Drosophila factor 2 and determined that it caused the release of the RNA component of RNA polymerase II elongation complexes in an ATP-dependent manner. We refer to factor 2 as a transcript release factor rather than a termination factor only because we have not determined if the polymerase is also released from the template. Since it is not likely that the polymerase continues to synthesize RNA after the transcript is released, it is probable that factor 2 acts as a true termination factor.
Factor 2 is involved in abortive elongation but is not completely responsible for the process. Abortive elongation is characterized by the rapid generation of short transcripts due to pausing of the polymerase followed by termination of some of the transcripts(13, 14) . Although factor 2 plays a role in transcript release, two of our results indicate that other factors also contribute to abortive elongation. First, the low salt washed complexes that were resistant to factor 2 were unable to synthesize long transcripts at 60 mM KCl even though the transcripts remained in elongation complexes (Fig. 4A). Second, complexes washed by high salt, though lacking factor 2, still synthesized shorter transcripts on average at 60 mM KCl compared with 250 mM KCl (Fig. 4B). It appeared that some of the abortive properties of the elongation complexes could be suppressed but not removed by high salt. Our earlier results (13, 14) and those presented here suggest that preinitiation complexes confer a negative elongation potential on the early elongation complexes. This could be due to a factor in preinitiation complexes, which remains in elongation complexes, or to the action of a factor on some component of elongation complexes, perhaps through a covalent modification.
Our results indicate that a fraction of early elongation complexes was resistant to transcript release. This was demonstrated by the inability of added factor 2 to cause additional transcript release from low salt washed complexes. If the salt remained low, the inhibitor remained associated with the complexes during elongation. The lack of function of factor 2 on isolated early elongation complexes (Fig. 3A) seemed to contradict the clear effect of added factor 2 in the continuous labeling transcription experiments using crude fractions (Fig. 2, B and E). This apparent discrepancy arose because the two assay systems were quite different. In the transcript release assay the effect of factor 2 was examined on washed elongation complexes, while in the continuous labeling assay the effect of factor 2 was tested in the presence of crude fractions without washing. Factors, including known elongation factors, in the continuous labeling assay might have influenced the activity of factor 2 or the inhibitor, and these factors would have been removed by the low salt wash. Elucidation of the properties of the inhibitor and identification of other factors that may be involved will be necessary to completely understand the function of factor 2 in elongation control.
The inhibitor of factor 2 function could be a nonspecific protein that blocks the association of factor 2 or could be a specific factor restricting the activity of factor 2 to a certain type of elongation complex. Partially purified factor 2 was shown to stimulate productive elongation by P-TEFb(15) . Preliminary results with pure factor 2 suggest the stimulatory effect was probably due to contaminating factors in the fraction (data not shown). In reactions containing the factors required for the generation of DRB-sensitive long runoff transcripts, the crude factor 2 fraction used did not inhibit the appearance of long transcripts(15) , suggesting that factor 2 selectively acted on a subset of elongation complexes. One intriguing possibility is that the inhibitor protects a subset of elongation complexes from factor 2 action and that these complexes are then acted upon by P-TEF to allow the transition into productive elongation. Alternatively, it is possible that only the complexes that factor 2 can act on are potentially productive. If this is the case, P-TEF would have to act before factor 2 caused transcript release. These opposing models could be tested by determining if P-TEF can act on the factor 2-resistant complexes.
The mechanism of factor 2 action may share some similarities with other termination factors. Factor 2 is like the E. coli rho factor and vaccinia capping enzyme in that it requires ATP for function(7, 17) . Unlike rho, however, factor 2 can associate with early elongation complexes containing RNA less than 10 nucleotides in length and cause transcript release. Such short transcripts are probably still sequestered within RNA polymerase II (18, 19) and are not accessible to RNA binding proteins. The tight interaction of factor 2 with early elongation complexes is more likely through DNA, RNA polymerase II itself, or other associated factors. A transcription/repair coupling factor (TRCF) has been identified in E. coli, which increases the rate of repair of transcribed regions(20, 21) . TRCF recognizes stalled elongation complexes caused by DNA lesions, nucleotide starvation, or protein roadblocks and dissociates the ternary complexes upon ATP hydrolysis. TRCF contains an RNA polymerase binding motif and binds double strand DNA but has little affinity for RNA. There is a possibility that factor 2 may be the eucaryotic homologue of E. coli TRCF, but protein sequence and detailed functional studies are needed to justify this hypothesis.