- Email: [email protected]
In a Head-on Collision, Two RNA Polymerases Approaching One Another on the Same DNA May Pass by One Another
doi:10.1016/j.jmb.2009.06.060
J. Mol. Biol. (2009) 391, 808–812
Available online at www.sciencedirect.com
COMMUNICATION
In a Head-on Collision, Two RNA Polymerases Approaching One Another on the Same DNA May Pass by One Another Na Ma 1,2,3 and William T. McAllister 1,4 ⁎ 1
Department of Microbiology and Immunology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA 2
Graduate Program in Molecular and Cellular Biology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA
Using a template that contains promoters for T3 and T7 RNA polymerases (RNAPs) in opposing orientations, and His-tagged derivatives of these RNAPs that allow immobilization on solid matrices, we have determined that a T7 elongation complex (EC) may be advanced past a halted T3 EC, and that after the collision the halted T3 EC may resume transcription. Since RNAPs moving in opposite directions use two different strands of the DNA as their templates, it seems likely that they manage to pass by one other by temporarily releasing their nontemplate strand while maintaining association with their template strand. © 2009 Elsevier Ltd. All rights reserved.
3
Department of Ophthalmology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA 4
Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 42 East Laurel Road, Stratford, NJ 08084, USA Received 27 March 2009; received in revised form 16 June 2009; accepted 23 June 2009 Available online 1 July 2009 Edited by R. Ebright
Keywords: Transcription; replication forks; template strand; RNA polymerase
During transcription, RNA polymerases (RNAPs) encounter many obstacles as they move along the template DNA. These include, for example, nucleo⁎Corresponding author. Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 42 East Laurel Road, Stratford, NJ 08084, USA. E-mail address: [email protected]. Abbreviations used: RNAP, RNA polymerase; EC, elongation complex; GTP, guanosine triphosphate; UTP, uridine triphosphate; dCTP, deoxycytidine triphosphate.
somes, repressors, replication forks, and other RNAPs. There are frequent occurrences of overlapping transcription units in which RNAPs may collide either in a codirectional manner (rear-end collision) or in an opposing manner (front-end collision). It is therefore of interest to determine how polymerases handle these situations. Earlier studies involving single-subunit and multisubunit bacterial RNAPs focused on the consequences of rear-end collisions. In the case of the multisubunit bacterial RNAPs, it was found that the trailing RNAP may facilitate the passage of the leading polymerase through a protein roadblock
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
Colliding Polymerases
and may also assist the leading polymerase to escape transient pausing (molecular cooperation).1–3 In the case of the single-subunit T7 RNAP, a rear-end collision was found to result in displacement of an elongation complex (EC) stalled downstream.4 To our knowledge, there have been no studies concerning the outcome of a head-on collision between two RNAPs. A T7 EC may be advanced past a halted T3 EC To allow two polymerases to move toward one another in a controlled manner, a template was constructed that contains a promoter for T7 RNAP and a promoter for T3 RNAP arranged in opposite directions (Fig. 1a). These two polymerases are highly specific and will not initiate at the heterologous promoter in the presence of their cognate promoter.13 The template is designed to allow T3 RNAP to form a stable EC halted at + 15 (T3 EC15) in the presence of guanosine triphosphate (GTP), UTP, [α- 32 P]ATP, and 3′-deoxycytidine triphosphate (dCTP) (Fig. 1b, step 1). The incorporation of 3′dCTP at the 3′ end of the T3 transcript acts as a chain terminator and prevents further extension of the RNA during subsequent steps. The use of a Histagged RNAP (H-T3 RNAP) allows subsequent immobilization of the complex on Ni2+–agarose beads.6,7 After a washing step to remove unbound
809 template and substrate, non-His-tagged T7 RNAP was loaded onto the same template in the presence of GTP and ATP, which allows T7 RNAP to form a complex halted at +15 (T7 EC15) (Fig. 1b, step 2). At this point the active sites of the two ECs are separated by 13 bp and the leading edges of the footprints of the complexes are expected to be separated by ∼5 bp (Fig. 1c). Note that the nonHis-tagged T7 EC will be retained on the beads only as a result of its assembly on a DNA template that is linked to the beads via the His-tagged T3 EC. This feature of the experimental design ensures that all T7 ECs examined are on templates that contain a halted T3 EC. The transcript in the T7 EC was then labeled by incorporation of [α-32P]UTP and the complex was advanced incrementally toward the halted T3 EC during subsequent cycles of washing and addition of different combinations of substrates. After each step of walking, samples were fractionated by brief centrifugation and the complexes remaining on the beads after washing or released during the wash were examined by gel electrophoresis (Fig. 1d). During walking, the active site of the T7 EC approaches that of the T3 EC and the distance between them diminishes until they overlap and then pass by one another (Fig. 1b, Dis). There was little dissociation of T7 or T3 ECs into the supernatant, and the fraction of T7 EC extended
Fig. 1. (a) A template that contains consensus promoters for T7 (red) and T3 (blue) RNAPs was formed by annealing together the oligonucleotides indicated (IDT DNA, Coralville, IA) at a concentration of 10 μM each in transcription buffer [20 mM Tris–acetate (pH 7.9), 10 mM Mg acetate, 0.1 mM EDTA, 0.05% Tween-20, 5 mM 2-mercaptoethanol).5 Numbered arrows above and below the sequence indicate the positions of the active sites of T7 and T3 ECs during each step of walking as described in (b) to (d). (b) Walking assays in which phage RNAPs having a hexahistidine affinity tag (His-tag) are immobilized on Ni2+–agarose beads and incrementally advanced by successive cycles of addition of appropriate combinations of substrate nucleotide triphosphates (NTPs) followed by washing were carried out as previously described.6,7 In step 1, T3 RNAP ECs halted at +15 were formed by incubating His-tagged T3 RNAP (H-T3RNAP; 1 μM final concentration) and template (1 μM final concentration) in the presence of 0.3 mM GTP (G), 0.1 mM [α-32P]ATP (A*; *indicates 32P label), 0.1 mM UTP (U), and 0.3 mM 3′-dCTP (3′dC). The complexes were immobilized on Ni2+–agarose beads and washed with transcription buffer. Samples of the wash and bead fractions were reserved for subsequent analysis. T7 RNAP without a His-tag was then added (1 μM final concentration) together with 0.3 mM GTP and 0.1 mM ATP, to allow formation of a T7 EC halted at +15 (T7 EC15, step 2). After washing to remove unbound T7 RNAP and substrates, [α-32P]UTP (U*) was added to allow T7 EC15 to be extended to + 16 (T7 EC16*; step 3). During subsequent steps, complexes were advanced by the addition of appropriate substrate as indicated (0.1 mM each NTP). The length of the transcript associated with the complexes at each step is indicated in the columns headed H-T3 EC or T7 EC; Dis indicates the distance (in base pairs) separating the active sites in the two complexes at each step (positive and negative numbers indicate distances before and after passing of the active sites, respectively). The efficiency of extension by the T7 EC during each step [calculated as (intensity of the T7 EC band at step n + 1)/(intensity of the band at step n)] is compared to that of a His-tagged T7 RNAP on the same template in the absence of a halted T3 EC8 in the last two columns. (c) The cartoon depicts the disposition of T7 EC 15 and H-T3 EC at the end of step 2; the leading and trailing edges of the T7 RNAP EC extend ∼ 19 bp upstream and 5–7 bp downstream of the active site.9–12 Dis, the distance separating the two active sites (yellow ovals); H, the presence of a His tag; the nascent transcript is indicated in green. (d). Samples of the wash and bead fractions reserved in each step were analyzed by electrophoresis in 20% (w/v) polyacrylamide gels containing 7 M urea; first lane in each pair, wash fraction, second lane, bead fraction. The identity of each transcript is noted. The minor band marked with an asterisk in step 3 reflects the formation of a labeled T3 EC14 complex that results from dissociation and reinitiation of T3 RNAP during steps 2 and 3, as it is extendable in the presence of CTP to a length of 20 nt in step 7 (as expected on the basis of the template sequence); the presence of this complex has no effect on the interpretation of the data. The minor band at 25 nt observed in steps 6–9 may result from low-level dissociation of T7 EC during step 6; however, a similar rate of release during this step is also observed in the absence of a halted T3 EC on the same template.8 (e) To confirm that the T7 transcripts observed in (d) were formed only on templates that were occupied by H-T3 RNAP (and hence retained by the beads) steps 1, 2, 3 and 9 were repeated in the absence or presence of H-T3 RNAP, as indicated. Labeled transcripts were observed only when H-T3 RNAP was present. (f) To determine if the T3 EC may be advanced after passage of the T7 EC, the experimental design was altered to include His-tagged T7 RNAP (H-T7 EC) and nontagged T3 RNAP (T3 EC) and walking was carried out in the presence of various combinations of NTPs, as indicated. (g) Samples of the wash and bead fractions reserved in each step in (f) were analyzed by electrophoresis in 20% (w/v) polyacrylamide gels.
810
Colliding Polymerases
during each step was similar to the level of extension observed on the same template in the absence of a halted T3 EC (Fig. 1b, compare last two columns). To exclude the possibility that some T7 EC might be retained on the beads through nonspecific interaction instead of through the His-tagged T3 EC, a control experiment was carried out in the absence of His-tagged T3 RNAP. As seen in Fig. 1e, no transcription activity was detected in the absence of H-T3 RNAP, indicating that the T7 products observed in Fig. 1d arise only by transcription of a template that has been retained on the beads via its occupancy by His-tagged T3 RNAP.
After the collision, the bypassed T3 EC may resume transcription Having shown that T7 EC can be walked past a halted T3 EC, we then asked what the fate of the T3 EC is after the T7 EC has passed by. The use of 3′dCTP as a chain terminator in the experimental scheme described above precluded subsequent extension of the T3 EC, and we therefore modified the experimental design (Fig. 1f and g). T7 EC15s were formed by incubating His-tagged T7 RNAP with template, GTP, and [α-32P]ATP, and the labeled complexes were immobilized on Ni 2+ –agarose beads. Non-his-tagged T3 RNAP was then loaded
Fig. 1 (legend on previous page)
811
Colliding Polymerases
onto the template in the presence of GTP and [α-32P]ATP, which allows the formation of a labeled T3 EC13. In the next step, GTP, ATP, and UTP were added to allow the advance of the T7 RNAP to position + 30 (T7 EC30) and the T3 EC to + 14 (T3 EC14). At this point, the active centers of the T3 and T7 complexes are juxtaposed. Some release of the T3 transcript was observed at this point, but most complexes remained associated with the beads and could be extended during subsequent steps (Fig. 1g). In summary, in this work we have determined that two phage RNAPs in opposing orientations may be incrementally advanced past each other, and that after the collision most complexes retain the ability to extend their transcripts. Since RNAPs moving in opposite directions use two different strands of the DNA as their templates, it seems likely that they manage to pass by one another by temporarily releasing their nontemplate strand while maintaining association with their template strand. This interpretation is consistent with structural studies in which it was observed that the template strand is sequestered within the EC, while the nontemplate strand is in a surface-exposed cleft.9,10 During the collision, there appears to be minimal destabilization of the transcription complexes, as the efficiency of extension of the T7 EC is about the same in the presence or absence of a halted T3 EC, even after extended washing of the immobilized complexes (six wash cycles, ∼ 2 min each; see Fig. 1b, last two columns). Additional experiments would be required to determine the extent of destabilization in a more quantitative manner. Separately, we attempted to determine if there was any pause or delay as a halted T7 EC was chased past a halted T3 EC, but were unable to detect any pausing under the limits of detection in these experiments (5 s; data not shown).8 The single-subunit phage RNAPs carry out all of the steps in the transcription cycle in the same manner as that of multisubunit RNAPs, but are structurally unrelated.14 It is therefore not clear whether the results reported here are generally applicable to other RNAPs. It has been shown, for example, that elongating phage RNAPs are less sensitive to bound proteins such as lac repressor and a noncleaving mutant of EcoRI than are bacterial enzymes.15–19 With the use of affinity-tagged multisubunit RNAPs and appropriately designed templates, it should be possible to determine what happens during a headon collision between two multisubunit RNAPs in the same manner as described here.
Acknowledgements This work was supported by NIH grant GM38147 to W.T.M. We are grateful to Drs. Dmitry Temiakov, Michael Anikin, and Richard Pomerantz for helpful comments. This work was submitted to the State
University of New York as partial fulfillment of the requirements for the doctoral degree of N.M.
References 1. Epshtein, V., Toulme, F., Rahmouni, A. R., Borukhov, S. & Nudler, E. (2003). Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727. 2. Epshtein, V. & Nudler, E. (2003). Cooperation between RNA polymerase molecules in transcription elongation. Science, 300, 801–805. 3. Wang, F. J. & Reiss, C. (1993). The collision of cotranscribing E. coli RNA polymerases studied in vitro. Biochem. Mol. Biol. Int. 30, 969–981. 4. Zhou, Y. & Martin, C. T. (2006). Observed instability of T7 RNA polymerase elongation complexes can be dominated by collision-induced “bumping”. J. Biol. Chem. 281, 24441–24448. 5. Jiang, M., Ma, N., Vassylyev, D. G. & McAllister, W. T. (2004). RNA displacement and resolution of the transcription bubble during transcription by T7 RNA polymerase. Mol. Cell, 15, 777–778. 6. Pomerantz, R. T., Ramjit, R., Gueroui, Z., Place, C., Anikin, M., Leuba, S. et al. (2005). A tightly regulated molecular motor based upon T7 RNA polymerase. Nano Lett. 5, 1698–1703. 7. Temiakov, D., Karasavas, P. E. & McAllister, W. T. (2000). Characterization of T7 RNA polymerase protein–DNA interactions during the initiation and elongation phases. In (Travers, A. A. & Buckle, M., eds), pp. 351–364, Oxford University Press, Oxford, UK. 8. Ma, N. (2005). RNA Displacement in Transcription by T7 RNAP. Doctoral thesis, State University of New York, Downstate Medical Center, Brooklyn, NY. 9. Yin, Y. W. & Steitz, T. A. (2002). Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science, 298, 1387–1395. 10. Tahirov, T. H., Temiakov, D., Anikin, M., Patlan, V., McAllister, W. T., Vassylyev, D. G. & Yokoyama, S. (2002). Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution. Nature, 420, 43–50. 11. Ikeda, R. A. & Richardson, C. C. (1986). Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. Proc. Natl. Acad. Sci. USA, 83, 3614–3618. 12. Huang, J. & Sousa, R. (2000). T7 RNA polymerase elongation complex structure and movement. J. Mol. Biol. 303, 347–358. 13. Klement, J. F., Moorefield, M. B., Jorgensen, E. D., Brown, J. E., Risman, S. & McAllister, W. T. (1990). Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site. J. Mol. Biol. 215, 21–29. 14. McAllister, W. T. (1997). Transcription by T7 RNA polymerase. Nucleic Acids Mol. Biol. 11, 15–25. 15. Pavco, P. A. & Steege, D. A. (1990). Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein. J. Biol. Chem. 265, 9960–9969. 16. Dubendorff, J. W. & Studier, F. W. (1991). Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J. Mol. Biol. 219, 61–68. 17. Giordano, T. J., Deuschle, U., Bujard, H. & McAllister, W. T. (1989). Regulation of coliphage T3 and T7 RNA
812 polymerases by the lac repressor–operator system. Gene, 84, 209–219. 18. Lopez, P. J., Guillerez, J., Sousa, R. & Dreyfus, M. (1998). On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor. J. Mol. Biol. 276, 861–875.
Colliding Polymerases
19. Pavco, P. A. & Steege, D. A. (1991). Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein. Nucleic Acids Res. 19, 4639–4646.
J. Mol. Biol. (2009) 391, 808–812
Available online at www.sciencedirect.com
COMMUNICATION
In a Head-on Collision, Two RNA Polymerases Approaching One Another on the Same DNA May Pass by One Another Na Ma 1,2,3 and William T. McAllister 1,4 ⁎ 1
Department of Microbiology and Immunology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA 2
Graduate Program in Molecular and Cellular Biology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA
Using a template that contains promoters for T3 and T7 RNA polymerases (RNAPs) in opposing orientations, and His-tagged derivatives of these RNAPs that allow immobilization on solid matrices, we have determined that a T7 elongation complex (EC) may be advanced past a halted T3 EC, and that after the collision the halted T3 EC may resume transcription. Since RNAPs moving in opposite directions use two different strands of the DNA as their templates, it seems likely that they manage to pass by one other by temporarily releasing their nontemplate strand while maintaining association with their template strand. © 2009 Elsevier Ltd. All rights reserved.
3
Department of Ophthalmology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA 4
Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 42 East Laurel Road, Stratford, NJ 08084, USA Received 27 March 2009; received in revised form 16 June 2009; accepted 23 June 2009 Available online 1 July 2009 Edited by R. Ebright
Keywords: Transcription; replication forks; template strand; RNA polymerase
During transcription, RNA polymerases (RNAPs) encounter many obstacles as they move along the template DNA. These include, for example, nucleo⁎Corresponding author. Department of Cell Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 42 East Laurel Road, Stratford, NJ 08084, USA. E-mail address: [email protected]. Abbreviations used: RNAP, RNA polymerase; EC, elongation complex; GTP, guanosine triphosphate; UTP, uridine triphosphate; dCTP, deoxycytidine triphosphate.
somes, repressors, replication forks, and other RNAPs. There are frequent occurrences of overlapping transcription units in which RNAPs may collide either in a codirectional manner (rear-end collision) or in an opposing manner (front-end collision). It is therefore of interest to determine how polymerases handle these situations. Earlier studies involving single-subunit and multisubunit bacterial RNAPs focused on the consequences of rear-end collisions. In the case of the multisubunit bacterial RNAPs, it was found that the trailing RNAP may facilitate the passage of the leading polymerase through a protein roadblock
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.
Colliding Polymerases
and may also assist the leading polymerase to escape transient pausing (molecular cooperation).1–3 In the case of the single-subunit T7 RNAP, a rear-end collision was found to result in displacement of an elongation complex (EC) stalled downstream.4 To our knowledge, there have been no studies concerning the outcome of a head-on collision between two RNAPs. A T7 EC may be advanced past a halted T3 EC To allow two polymerases to move toward one another in a controlled manner, a template was constructed that contains a promoter for T7 RNAP and a promoter for T3 RNAP arranged in opposite directions (Fig. 1a). These two polymerases are highly specific and will not initiate at the heterologous promoter in the presence of their cognate promoter.13 The template is designed to allow T3 RNAP to form a stable EC halted at + 15 (T3 EC15) in the presence of guanosine triphosphate (GTP), UTP, [α- 32 P]ATP, and 3′-deoxycytidine triphosphate (dCTP) (Fig. 1b, step 1). The incorporation of 3′dCTP at the 3′ end of the T3 transcript acts as a chain terminator and prevents further extension of the RNA during subsequent steps. The use of a Histagged RNAP (H-T3 RNAP) allows subsequent immobilization of the complex on Ni2+–agarose beads.6,7 After a washing step to remove unbound
809 template and substrate, non-His-tagged T7 RNAP was loaded onto the same template in the presence of GTP and ATP, which allows T7 RNAP to form a complex halted at +15 (T7 EC15) (Fig. 1b, step 2). At this point the active sites of the two ECs are separated by 13 bp and the leading edges of the footprints of the complexes are expected to be separated by ∼5 bp (Fig. 1c). Note that the nonHis-tagged T7 EC will be retained on the beads only as a result of its assembly on a DNA template that is linked to the beads via the His-tagged T3 EC. This feature of the experimental design ensures that all T7 ECs examined are on templates that contain a halted T3 EC. The transcript in the T7 EC was then labeled by incorporation of [α-32P]UTP and the complex was advanced incrementally toward the halted T3 EC during subsequent cycles of washing and addition of different combinations of substrates. After each step of walking, samples were fractionated by brief centrifugation and the complexes remaining on the beads after washing or released during the wash were examined by gel electrophoresis (Fig. 1d). During walking, the active site of the T7 EC approaches that of the T3 EC and the distance between them diminishes until they overlap and then pass by one another (Fig. 1b, Dis). There was little dissociation of T7 or T3 ECs into the supernatant, and the fraction of T7 EC extended
Fig. 1. (a) A template that contains consensus promoters for T7 (red) and T3 (blue) RNAPs was formed by annealing together the oligonucleotides indicated (IDT DNA, Coralville, IA) at a concentration of 10 μM each in transcription buffer [20 mM Tris–acetate (pH 7.9), 10 mM Mg acetate, 0.1 mM EDTA, 0.05% Tween-20, 5 mM 2-mercaptoethanol).5 Numbered arrows above and below the sequence indicate the positions of the active sites of T7 and T3 ECs during each step of walking as described in (b) to (d). (b) Walking assays in which phage RNAPs having a hexahistidine affinity tag (His-tag) are immobilized on Ni2+–agarose beads and incrementally advanced by successive cycles of addition of appropriate combinations of substrate nucleotide triphosphates (NTPs) followed by washing were carried out as previously described.6,7 In step 1, T3 RNAP ECs halted at +15 were formed by incubating His-tagged T3 RNAP (H-T3RNAP; 1 μM final concentration) and template (1 μM final concentration) in the presence of 0.3 mM GTP (G), 0.1 mM [α-32P]ATP (A*; *indicates 32P label), 0.1 mM UTP (U), and 0.3 mM 3′-dCTP (3′dC). The complexes were immobilized on Ni2+–agarose beads and washed with transcription buffer. Samples of the wash and bead fractions were reserved for subsequent analysis. T7 RNAP without a His-tag was then added (1 μM final concentration) together with 0.3 mM GTP and 0.1 mM ATP, to allow formation of a T7 EC halted at +15 (T7 EC15, step 2). After washing to remove unbound T7 RNAP and substrates, [α-32P]UTP (U*) was added to allow T7 EC15 to be extended to + 16 (T7 EC16*; step 3). During subsequent steps, complexes were advanced by the addition of appropriate substrate as indicated (0.1 mM each NTP). The length of the transcript associated with the complexes at each step is indicated in the columns headed H-T3 EC or T7 EC; Dis indicates the distance (in base pairs) separating the active sites in the two complexes at each step (positive and negative numbers indicate distances before and after passing of the active sites, respectively). The efficiency of extension by the T7 EC during each step [calculated as (intensity of the T7 EC band at step n + 1)/(intensity of the band at step n)] is compared to that of a His-tagged T7 RNAP on the same template in the absence of a halted T3 EC8 in the last two columns. (c) The cartoon depicts the disposition of T7 EC 15 and H-T3 EC at the end of step 2; the leading and trailing edges of the T7 RNAP EC extend ∼ 19 bp upstream and 5–7 bp downstream of the active site.9–12 Dis, the distance separating the two active sites (yellow ovals); H, the presence of a His tag; the nascent transcript is indicated in green. (d). Samples of the wash and bead fractions reserved in each step were analyzed by electrophoresis in 20% (w/v) polyacrylamide gels containing 7 M urea; first lane in each pair, wash fraction, second lane, bead fraction. The identity of each transcript is noted. The minor band marked with an asterisk in step 3 reflects the formation of a labeled T3 EC14 complex that results from dissociation and reinitiation of T3 RNAP during steps 2 and 3, as it is extendable in the presence of CTP to a length of 20 nt in step 7 (as expected on the basis of the template sequence); the presence of this complex has no effect on the interpretation of the data. The minor band at 25 nt observed in steps 6–9 may result from low-level dissociation of T7 EC during step 6; however, a similar rate of release during this step is also observed in the absence of a halted T3 EC on the same template.8 (e) To confirm that the T7 transcripts observed in (d) were formed only on templates that were occupied by H-T3 RNAP (and hence retained by the beads) steps 1, 2, 3 and 9 were repeated in the absence or presence of H-T3 RNAP, as indicated. Labeled transcripts were observed only when H-T3 RNAP was present. (f) To determine if the T3 EC may be advanced after passage of the T7 EC, the experimental design was altered to include His-tagged T7 RNAP (H-T7 EC) and nontagged T3 RNAP (T3 EC) and walking was carried out in the presence of various combinations of NTPs, as indicated. (g) Samples of the wash and bead fractions reserved in each step in (f) were analyzed by electrophoresis in 20% (w/v) polyacrylamide gels.
810
Colliding Polymerases
during each step was similar to the level of extension observed on the same template in the absence of a halted T3 EC (Fig. 1b, compare last two columns). To exclude the possibility that some T7 EC might be retained on the beads through nonspecific interaction instead of through the His-tagged T3 EC, a control experiment was carried out in the absence of His-tagged T3 RNAP. As seen in Fig. 1e, no transcription activity was detected in the absence of H-T3 RNAP, indicating that the T7 products observed in Fig. 1d arise only by transcription of a template that has been retained on the beads via its occupancy by His-tagged T3 RNAP.
After the collision, the bypassed T3 EC may resume transcription Having shown that T7 EC can be walked past a halted T3 EC, we then asked what the fate of the T3 EC is after the T7 EC has passed by. The use of 3′dCTP as a chain terminator in the experimental scheme described above precluded subsequent extension of the T3 EC, and we therefore modified the experimental design (Fig. 1f and g). T7 EC15s were formed by incubating His-tagged T7 RNAP with template, GTP, and [α-32P]ATP, and the labeled complexes were immobilized on Ni 2+ –agarose beads. Non-his-tagged T3 RNAP was then loaded
Fig. 1 (legend on previous page)
811
Colliding Polymerases
onto the template in the presence of GTP and [α-32P]ATP, which allows the formation of a labeled T3 EC13. In the next step, GTP, ATP, and UTP were added to allow the advance of the T7 RNAP to position + 30 (T7 EC30) and the T3 EC to + 14 (T3 EC14). At this point, the active centers of the T3 and T7 complexes are juxtaposed. Some release of the T3 transcript was observed at this point, but most complexes remained associated with the beads and could be extended during subsequent steps (Fig. 1g). In summary, in this work we have determined that two phage RNAPs in opposing orientations may be incrementally advanced past each other, and that after the collision most complexes retain the ability to extend their transcripts. Since RNAPs moving in opposite directions use two different strands of the DNA as their templates, it seems likely that they manage to pass by one another by temporarily releasing their nontemplate strand while maintaining association with their template strand. This interpretation is consistent with structural studies in which it was observed that the template strand is sequestered within the EC, while the nontemplate strand is in a surface-exposed cleft.9,10 During the collision, there appears to be minimal destabilization of the transcription complexes, as the efficiency of extension of the T7 EC is about the same in the presence or absence of a halted T3 EC, even after extended washing of the immobilized complexes (six wash cycles, ∼ 2 min each; see Fig. 1b, last two columns). Additional experiments would be required to determine the extent of destabilization in a more quantitative manner. Separately, we attempted to determine if there was any pause or delay as a halted T7 EC was chased past a halted T3 EC, but were unable to detect any pausing under the limits of detection in these experiments (5 s; data not shown).8 The single-subunit phage RNAPs carry out all of the steps in the transcription cycle in the same manner as that of multisubunit RNAPs, but are structurally unrelated.14 It is therefore not clear whether the results reported here are generally applicable to other RNAPs. It has been shown, for example, that elongating phage RNAPs are less sensitive to bound proteins such as lac repressor and a noncleaving mutant of EcoRI than are bacterial enzymes.15–19 With the use of affinity-tagged multisubunit RNAPs and appropriately designed templates, it should be possible to determine what happens during a headon collision between two multisubunit RNAPs in the same manner as described here.
Acknowledgements This work was supported by NIH grant GM38147 to W.T.M. We are grateful to Drs. Dmitry Temiakov, Michael Anikin, and Richard Pomerantz for helpful comments. This work was submitted to the State
University of New York as partial fulfillment of the requirements for the doctoral degree of N.M.
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