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Address correspondence to Peter R. Cook, Sir William Dunn School of Pathology, South Parks Rd., Oxford OX1 3RE, UK. Tel.: 44-1865-275528. Fax: 44-1865-275515. E-mail: peter.cook{at}path.ox.ac.uk
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Abstract |
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Key Words: green fluorescent protein; photobleaching; actinomycin D; DRB; kinetics
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Introduction |
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The largest catalytic subunit of the polymerase bears a temperature-sensitive mutation in the CHO cell line, tsTM4. The wild-type subunit from human cells was tagged with GFP and expressed in tsTM4; this construct complemented the defect at the restrictive temperature, and enabled the mutant cells to grow normally (Sugaya et al., 2000). This indicates that the tagged polymerase must be functional. However, these cells contain both endogenous and tagged polymerases, and we can estimate their relative contributions to the total polymerizing activity as follows: during elongation, the COOH-terminal domain of the largest catalytic subunit becomes hyperphosphorylated and reactive to the H5 antibody. As a result, this hyperphosphorylated form (form IIO) is widely used as a marker for the active enzyme (Dahmus, 1996; Komarnitsky et al., 2000). Under the growth conditions used here, immunoblotting reveals that most of the H5-reactive form in the cell is the GFP-polymerase (GFP-pol) rather than the endogenous enzyme (Sugaya et al., 2000). We now use these cells to analyze the mobility of the GFP-pol, concentrating on changes occurring over the minutes required to complete a transcription cycle (including initiation, elongation, and termination). Determining whether GFP-pol diffuses as a core enzyme of 500 kD or a larger complex of 1,0002,000 kD (Lee and Young, 2000) requires analysis over fractions of a second and the development of fluorescent standards of appropriate size. However, note that no larger complexes involved in repair have been detected (Houtsmuller et al., 1999). The kinetics are consistent with
75% of the GFP-pol being able to move quickly, with the remainder being transiently immobile (association t1/2
20 min). No fraction immobilized in an inactive preinitiation complex could be detected. We also used a conventional biochemical approach (involving radiolabeling nascent transcripts with [3H]uridine) to confirm that the endogenous enzyme in wild-type cells completes a transcription cycle with roughly similar kinetics. Using current estimates of the length of a typical gene and the rate of elongation, we calculate that a polymerase would be engaged for only one half to five sixths of a transcription cycle; then, a typical expressed transcription unit would actually be transcribed for only a minority of the time.
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Results |
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Kinetics in wild-type cells determined by radiolabeling
We also examined the kinetics of the endogenous polymerase in wild-type cells using an independent technique (Fig. 2). Parental CHO-K1 cells were encapsulated in agarose beads to protect them, grown in [3H]uridine for different times, treated without and with sarkosyl, and the amount of radioactivity in RNA counted. Sarkosyl is a strong detergent that is widely used to extract completed transcripts while leaving nascent ones still associated with the polymerase engaged on its template (Kovelman and Roeder, 1990; Szentirmay and Sawadogo, 1994). Under our conditions, it removes completed transcripts, but leaves nascent ones in beads (Jackson et al., 1998; Kimura et al., 1999; Sugaya et al., 2000). In principle, radioactivity in all transcripts should increase, whereas that in nascent transcripts rises to a plateau (Fig. 2 A). In practice, there is a short lag of 2.5 min as internal pools become labeled; then, the maximum rate of incorporation is reached before turnover tempers the increase (Fig. 2 B, curve 1). Nascent (sarkosyl-resistant) transcripts initially become labeled with similar kinetics, but soon become saturated (Fig. 2 B, curve 2). Excluding the initial lag, it takes 14 min for label in nascent transcripts to reach half the maximum. As the increase in labeling is determined by rates of initiation and elongation, and as initiation and termination rates must be equal, this half-time is that of a complete transcription cycle. These results confirm those obtained by FRAP, and indicate that tagged and wild-type enzymes behave similarly.
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Discussion |
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Two properties of the tagged polymerase were analyzed in detail: (1) the engaged fraction; and (2) the time taken to complete half a transcription cycle. Given the successful genetic complementation, we might expect (but cannot formally prove) that the tagged polymerase behaves normally, simply because it is difficult to imagine how the cells could survive if the properties of such an important enzyme were changed significantly. Fortunately, the engaged fraction of the tagged polymerase in the nucleus (Fig. 1, B and E; see Materials and methods) turns out to be similar to that found previously for the untagged enzyme in HeLa cells (Kimura et al., 1999). Moreover, the half-life of GFP-pol in the complemented cells also proves to be similar to that of the untagged enzyme in wild-type CHO cells (Fig. 2). Taken at face value, this suggests that any overexpression has little influence on nuclear kinetics, and there is a simple explanation of why this may be so. The other subunits in the core enzyme will be expressed at the usual levels, and so excess GFP-tagged subunits cannot be incorporated into the core enzyme; therefore, we might expect them to remain in the cytoplasm, and this seems to be the case (Sugaya et al., 2000). But as with all studies using GFP as a tag, it remains possible that the tag and/or any overexpression influences the kinetics seen by FRAP and FLIP, and this should be borne in mind during the following discussion. Note that we did not study the diffusion of the GFP-pol for the reasons discussed in the Introduction, and because its apparent diffusion rate turns out to be sufficiently fast that it makes essentially no contribution to the slow kinetics analyzed (Fig. 1 E). However, the fast fraction appears to diffuse more slowly than GFP alone (Fig. 1 E; unpublished data), and future studies are needed to determine whether this reflects a larger size (perhaps of a core or holoenzyme) and/or repeated association and dissociation from different nuclear binding sites (e.g., factories, speckles, or Cajal bodies).
The kinetics of the polymerase
We can draw several conclusions from this work. First, results from both FRAP and FLIP (Fig. 1, B and E) are consistent with 2025% GFP-pol being engaged. Biochemical analysis shows that a similar fraction of the untagged polymerase is engaged in HeLa cells (Kimura et al., 1999), but, as discussed above, this similarity could arise fortuitously. Second, little transcriptionally inactive GFP-pol is found in stable complexes, as DRB eliminates almost all the slow (engaged) fraction (Fig. 1 B). This result is not supported by additional data on the natural polymerase, but it implies that any unengaged enzyme on promoters or in larger structures like holoenzymes, preinitiation complexes, or factories (McCracken et al., 1997; Cook, 1999; Lee and Young, 2000) must exchange rapidly with soluble molecules (Misteli, 2001).
The third result surprised us. Data obtained using FRAP (on the tagged polymerase) and radiolabeling (on the natural enzyme) indicate that it takes 1420 min to complete half a transcription cycle (Figs. 1 B and 2 B). How does this compare with other estimates (Jackson et al., 2000)? Although several highly active (polymerase II) transcription units like those encoding a heat shock protein (Lis and Wu, 1993), globin (Wijgerde et al., 1995), and actin (Femino et al., 1998) have been studied, no in vivo data exist for the time taken to initiate and terminate on a "typical" unit. Therefore, we determined it as follows: first, we calculated the average elongation time from the length of a transcription unit and the polymerization rate. Assuming a transcription unit in a CHO cell has the same length as a human gene (median length 14 kbp; International Human Genome Sequencing Consortium, 2001), and a polymerization rate of 1.12.5 x 103 nucleotides/min (Jackson et al., 2000), a typical transcription unit would be copied in 613 min. This complete elongation time is less than the half-times obtained by FRAP or radiolabeling, so elongating a typical gene probably occupies less than one half to one sixth of the transcription cycle. This fraction roughly equals that of all molecules that are transiently immobilized (i.e., one quarter to one fifth), as might be expected under steady-state conditions. These results are also consistent with the widely held assumption that initiation is rate-limiting. It follows that more than one engaged polymerase would rarely be found on a typical (expressed) transcription unit. This conclusion runs counter to the widespread belief that most polymerase II units are associated with many engaged enzymes. Although active units like the chorion and hsp70 genes in Drosophila, and the globin and actin genes in mammals can be associated with many polymerases (Osheim et al., 1985; O'Brien and Lis, 1991; Wijgerde et al., 1995; Femino et al., 1998), studies of typical (expressed) genes show that they are associated with one or less polymerase (Laird and Chooi, 1976; Fakan et al., 1986; Jackson et al., 1998; for review see Jackson et al., 2000). For example, the Drosophila genes encoding two household genes (tubulin B1 and glyceraldehyde phosphate dehydrogenase) are typically associated with <1 polymerase (Rougvie and Lis, 1990), and the mammalian actin gene under steady-state conditions with
2 (Femino et al., 1998). Even the major late unit of adenovirus, which is widely believed to be one of the most active units in a mammalian cell, has only one polymerase approximately every 7.5 kbp (Beyer et al., 1981; Wolgemuth and Hsu, 1981). These low numbers contrast with the
125 polymerases/transcripts seen on an active polymerase I unit (Miller, 1981).
Finally, actD decreases the fast fraction seen by FRAP (Fig. 3 A, curve 2) and FLIP (Fig. 3 B, curve 2), suggesting the polymerase stalls at the intercalated drug as continuing initiation increases the engaged fraction. Although this is unsupported by independent data on the natural enzyme, we have no reason to believe that the GFP tag influences the way the inhibitor acts.
Models for the initiation of transcription
Taken at face value, these data constrain current models for initiation. For example, the stepwise assembly model sees TBP binding to a promoter, followed successively by transcription factor (TF) IIB, polymerase/TFIIF, TFIIE, and TFIIH (Orphanides et al., 1996). However, little TFIIB-GFP (Chen et al., 2002) or unengaged GFP-pol is immobilized (Fig. 1), so assembly must be rapid relative to the other phases in the cycle. Another model sees the polymerase assembled into a stable transcription complex that repeatedly transcribes the same gene (Brown, 1984), but then the half-life obtained by radiolabeling should be much less than that obtained by FRAP. Note that our results do not exclude the possibility that a stable "scaffold" containing TFs facilitates recycling (Yudkovsky et al., 2000), but that scaffold would have to be free of polymerase.
Our data are consistent with the following parsimonious model for the transcription of a typical unit (Fig. 4). During most of the cycle, the polymerase, TFs, and promoter collide at random to bind and dissociate immediately. Only occasionally do collisions occur in the appropriate sequence that permit rapid assembly of a preinitiation complex (probably in the order described above), and this complex soon initiates. Next, elongation occupies one quarter of the cycle before the polymerase terminates and dissociates quickly. It follows that a typical "active" (i.e., expressed) transcription unit is misnamed, as it spends a significant time not being transcribed. Note that many newly made transcripts are nongenic, and it remains to be seen whether the transcription cycle of genic transcription units is shorter than that of nongenic units.
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Materials and methods |
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Transcriptional activity of bleached regions
The transcriptional activity of bleached regions of cells was assessed as follows: C23 cells grown on a glass-bottomed dish marked with a grid (MatTek) were bleached as for FRAP, incubated for 1070 min, permeabilized, nascent transcripts were extended in Br-UTP, fixed, and immunolabeled (Pombo et al., 1999; Sugaya et al., 2000) using mouse anti-BrdU (1/200; Caltag) and donkey Cy3-conjugated antimouse immunoglobulin (1/200; Jackson ImmunoResearch Laboratories). Bleached cells were identified using the grid, and green and red confocal images were collected sequentially (4% power with Ar laser and 515/528 nm emission filter; 5% power with 1-mW HeNe laser and 560LP filter). The distribution and intensity of sites containing Br-RNA was similar in bleached and unbleached areas.
Radiolabeling
Wild-type CHO-K1 cells expressing only the endogenous polymerase were grown overnight in 1.85 kBq/ml methyl-[14C]thymidine to label DNA uniformly, encapsulated (Jackson and Cook, 1985) in agarose beads (107 cells/ml bead), regrown (4 h), and incubated in 3.7 Mbq/ml [3H]uridine. Incorporation was stopped by transferring 1-ml samples to 14 ml ice-cold PBS; beads were pelleted (3,000 rpm; 5 min; 4°C), washed two times in PBS, and radioactivity was measured by scintillation counting (Jackson and Cook, 1985). In some cases, beads were washed (5 min) in ice-cold 0.5% sarkosyl, spun, left overnight in ice-cold 0.5% sarkosyl, and washed two times before measuring radioactivity. 3H counts (in RNA) were normalized using 14C counts (in DNA).
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Footnotes |
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K. Sugaya's present address is Research Center for Radiation Safety, National Institute of Radiological Sciences, 4-9-1, Anagawa, Chiba 263-8555 Japan.
* Abbreviations used in this paper: actD, actinomycin D; DRB, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole; FLIP, fluorescence loss in photobleaching; GFP-pol, GFP-polymerase; TF, transcription factor.
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Acknowledgments |
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Submitted: 4 June 2002
Revised: 22 October 2002
Accepted: 23 October 2002
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References |
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