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Creative Math of RNA Polymerase III Termination: Sense Plus Antisense Makes More Sense
Molecular Cell
Previews Creative Math of RNA Polymerase III Termination: Sense Plus Antisense Makes More Sense Irina Artsimovitch1,* and Georgi A. Belogurov2 1Department
of Microbiology and Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA of Biochemistry, University of Turku, Turku 20014, Finland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.06.003 2Department
In this issue of Molecular Cell, Arimbasseri and Maraia (2015) demonstrate that yeast RNA polymerase III integrates inputs from both strands of the DNA template and three dedicated protein subunits to trigger the highly controlled release of the nascent RNA transcript. Transcription termination is essential to maintain gene boundaries, prevent synthesis of interfering antisense RNAs, and release RNA polymerase (RNAP) from its template. Although all RNAPs rely on termination control mechanisms to determine the position of RNA release, for none is the precise control of termination more important than for RNAPIII, the enzyme that synthesizes a variety of short RNA species, including tRNAs, 5S rRNA, and U6 snRNA. In rapidly growing yeast, RNAPIII makes several million RNA molecules per generation, producing and releasing transcripts at a rate of 1/s (Dieci et al., 2013). This astonishingly high rate is attributable to facilitated reinitiation, wherein following the first slow round of promoter complex formation, RNAPIII rapidly reloads onto the same template multiple times. The native termination signal and RNAPIII-specific subunits C11, C37, and C53 are crucial for facilitated reinitiation (Dieci et al., 2013). Yet, despite this regulatory complexity, RNAPIII requires only a simple termination signal composed of 4–6 thymidine residues in the coding strand and no auxiliary factors. In this issue of Molecular Cell, Arimbasseri and Maraia (2015) show that this bare-bones signal is surprisingly multifaceted, with the contributions from both DNA strands integrated to induce RNA release at the correct position in response to C11, C37, and C53. Arimbasseri and Maraia compare the termination properties of a complete 17subunit RNAPIII and variants that either lack C53, C37, and C11 (RNAPIIID) or have altered versions thereof. C37 and C53 are distant homologs of the RNAPII
initiation factor TFIIF, whereas C11 is a distant homolog of the RNAPII subunit Rpb9 and transcript cleavage factor TFIIS. The authors assemble RNAPIII transcription elongation complexes (TECs) on synthetic nucleic acid scaffolds containing nine consecutive adenines in the template DNA strand. Complete RNAPIII pauses upon entry into A-track (mainly at U4, the last nucleotide added to the RNA chain) and releases U5-U8 RNAs, whereas RNAPIIID fails to pause and releases RNA only at the end of A-track (mainly at U8–U9). Since typical RNAPIII terminators contain fewer than seven consecutive adenines, RNA release at U5–U7 (proximal terminator) likely represents the physiologically relevant mechanism. A set of elegant experiments with variant scaffolds revealed the unexpected role of the non-template DNA in termination. The A9-track appears to be sufficient to commit the complete and RNAPIIID to termination as RNA was released at U8–U9 even when the nontemplate strand was altered (Figure 1A). However, termination at the correct position is critical for reinitiation (Dieci et al., 2013), and release at U8–U9 may be irrelevant. The authors show that deletion of four amino acid residues in C37 or substitutions of the first four non-template thymidines abrogated pausing and RNA release at the ‘‘relevant’’ proximal terminator, whereas a substitution of the fifth thymidine retained pausing but abolished RNA release. These results provide a molecular explanation for earlier observations that C37/C53 were necessary for RNAPIII pausing within the terminator (Landrieux et al., 2006) and support a
974 Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc.
model in which sequence-specific interactions between C37 and the non-template DNA mediate the formation of a metastable pre-termination complex (PTC) (Figure 1B). In the PTC, several components collaborate to induce RNA release and enable productive reinitiation. The template strand A-track slows RNAPIII down and destabilizes the TEC by forming an unstable hybrid with the U-track in the nascent RNA. The non-template strand engages the C37/53 complex, additionally slowing RNAP. RNAPIII is prone to backtrack while transcribing through A-tracks, making it susceptible to C11, which rescues arrested TECs through cleavage of the nascent RNA. C37/53 and C11 (but not its RNA cleavage activity) are necessary for facilitated reinitiation. C37/53 and C11 may mediate conformational changes into a termination/reinitiation state—they form a complex located near the front edge of the moving RNAP and contain domains that approach the RNAPIII active site (Figure 1C). In addition, C37/53 interacts with the initiation factor TFIIIB, which remains bound to the promoter after initiation (Dieci et al., 2013). What are the structural changes that accompany the PTC formation? A T-track in the coding strand is part of the termination signal in bacteria (Gusarov and Nudler, 1999), archaea (Santangelo and Reeve, 2006), and RNAPIII, suggesting that termination could be mechanistically similar in these systems. In bacteria, the 9–10 bp RNA:DNA hybrid characteristic for an active TEC is thought to shorten to %7 bp upon invasion of the terminator RNA hairpin (Peters et al., 2011). We
Molecular Cell
Previews
Figure 1. Formation of the PTC (A) The T-track in the non-template strand and C37 are required for pausing and termination in the ‘‘correct,’’ proximal region, as shown by Arimbasseri and Maraia (2015). (B) The RNAP (gray oval) PTC is stabilized by interactions of the non-template DNA strand with C37 (orange) or by formation of the nascent RNA hairpin. (C) The proposed termination/reinitiation pathway. Following TFIIIB (green) and TFIIIC (not shown)-mediated recruitment, RNAPIII initiates at the start site (a bent arrow) and transcribes until it reaches the T-track. Domains of C11 (yellow), C37, and C53 (pink) extend toward the RNAP active site (a red dot). Interactions between the non-template T residues and C37 mediate pausing (at U4) and formation of a metastable PTC. Addition of subsequent UMP residues triggers further destabilization of the hybrid and RNA release. RNAP rebinds the promoter via interactions between C37 and TFIIIB, which remains associated with the promoter, whereas TFIIIC is dispensable for recycling on most genes. DNA wrapping could provide easy means for RNAP III handover. Alternatively, RNAPIII could slide back to the transcription start, only 100 nucleotides away on tRNA genes; however, the reported high occupancy of RNAPIII makes this scenario less likely (see Dieci et al., 2013 for a review). The conformation and occupancy of the transcribed DNA are yet unknown, as indicated by breaks therein.
propose that formation of the RNAPIII PTC also involves shortening of the RNA:DNA hybrid from the upstream side to 6–7 bp. Although in some species RNAPIII can terminate on as few as 4 Ts, 6 Ts appear to work in all cases. In a bacterial PTC, the shortened hybrid state is stabilized by the nascent RNA hairpin, whereas in RNAPIII PTC interactions of C37 with the non-template DNA may play a similar role (Figure 1B). It is noteworthy that the template DNA conformation in a bacterial initiation complex structure suggests that only a shorter 6–7 bp hybrid can form (Zuo and Steitz, 2015).
Thus, upon shortening of the RNA:DNA hybrid, the resulting PTC may resemble the initiation complex. The shortened U-rich hybrid would allow for RNA release from the enzyme, which could then rebind the promoter (Figure 1C). Isomerization into the PTC is likely mediated by largescale motions of the core subunits, such as ratcheting (Tagami et al., 2010), and the PTC may be additionally stabilized by RNAPIII initiation factors. Shortening of the RNA:DNA hybrid as the major transition accompanying the PTC formation provides an attractive explanation for the link between termination and reinitiation.
The sense or coding strand is commonly referred to as the non-template strand, an arguably disparaging term reflecting the fact that the coding strand is non-essential for transcription in vitro and is far less involved in RNA synthesis than the template strand. However, the non-template strand has long been known to play a leading role during initiation, where it is directly recognized by RNAP and accessory factors. Recent data indicate that interactions between the non-template DNA and the elongating RNAP (Vvedenskaya et al., 2014) or transcription factors (Yakhnin and Babitzke,
Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc. 975
Molecular Cell
Previews 2014) control RNA chain elongation. The report by Arimbasseri and Maraia provides compelling evidence for the importance of sequence-specific recognition of the non-template DNA during termination by yeast RNAPIII. Thus, besides encoding the primary sequences of noncoding RNAs and proteins indirectly, the non-template strand often carries the regulatory instructions that are recognized directly by diverse components of the transcription machinery during all stages of transcription.
REFERENCES
Santangelo, T.J., and Reeve, J.N. (2006). J. Mol. Biol. 355, 196–210.
Arimbasseri, A.G., and Maraia, R.J. (2015). Mol. Cell 58, this issue, 1124–1132.
Tagami, S., Sekine, S., Kumarevel, T., Hino, N., Murayama, Y., Kamegamori, S., Yamamoto, M., Sakamoto, K., and Yokoyama, S. (2010). Nature 468, 978–982.
Dieci, G., Bosio, M.C., Fermi, B., and Ferrari, R. (2013). Biochim. Biophys. Acta 1829, 331–341. Gusarov, I., and Nudler, E. (1999). Mol. Cell 3, 495–504. Landrieux, E., Alic, N., Ducrot, C., Acker, J., Riva, M., and Carles, C. (2006). EMBO J. 25, 118–128. Peters, J.M., Vangeloff, A.D., and Landick, R. (2011). J. Mol. Biol. 412, 793–813.
976 Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc.
Vvedenskaya, I.O., Vahedian-Movahed, H., Bird, J.G., Knoblauch, J.G., Goldman, S.R., Zhang, Y., Ebright, R.H., and Nickels, B.E. (2014). Science 344, 1285–1289. Yakhnin, A.V., and Babitzke, P. (2014). Curr. Opin. Microbiol. 18, 68–71. Zuo, Y., and Steitz, T.A. (2015). Mol. Cell 58, 534–540.
Previews Creative Math of RNA Polymerase III Termination: Sense Plus Antisense Makes More Sense Irina Artsimovitch1,* and Georgi A. Belogurov2 1Department
of Microbiology and Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA of Biochemistry, University of Turku, Turku 20014, Finland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.06.003 2Department
In this issue of Molecular Cell, Arimbasseri and Maraia (2015) demonstrate that yeast RNA polymerase III integrates inputs from both strands of the DNA template and three dedicated protein subunits to trigger the highly controlled release of the nascent RNA transcript. Transcription termination is essential to maintain gene boundaries, prevent synthesis of interfering antisense RNAs, and release RNA polymerase (RNAP) from its template. Although all RNAPs rely on termination control mechanisms to determine the position of RNA release, for none is the precise control of termination more important than for RNAPIII, the enzyme that synthesizes a variety of short RNA species, including tRNAs, 5S rRNA, and U6 snRNA. In rapidly growing yeast, RNAPIII makes several million RNA molecules per generation, producing and releasing transcripts at a rate of 1/s (Dieci et al., 2013). This astonishingly high rate is attributable to facilitated reinitiation, wherein following the first slow round of promoter complex formation, RNAPIII rapidly reloads onto the same template multiple times. The native termination signal and RNAPIII-specific subunits C11, C37, and C53 are crucial for facilitated reinitiation (Dieci et al., 2013). Yet, despite this regulatory complexity, RNAPIII requires only a simple termination signal composed of 4–6 thymidine residues in the coding strand and no auxiliary factors. In this issue of Molecular Cell, Arimbasseri and Maraia (2015) show that this bare-bones signal is surprisingly multifaceted, with the contributions from both DNA strands integrated to induce RNA release at the correct position in response to C11, C37, and C53. Arimbasseri and Maraia compare the termination properties of a complete 17subunit RNAPIII and variants that either lack C53, C37, and C11 (RNAPIIID) or have altered versions thereof. C37 and C53 are distant homologs of the RNAPII
initiation factor TFIIF, whereas C11 is a distant homolog of the RNAPII subunit Rpb9 and transcript cleavage factor TFIIS. The authors assemble RNAPIII transcription elongation complexes (TECs) on synthetic nucleic acid scaffolds containing nine consecutive adenines in the template DNA strand. Complete RNAPIII pauses upon entry into A-track (mainly at U4, the last nucleotide added to the RNA chain) and releases U5-U8 RNAs, whereas RNAPIIID fails to pause and releases RNA only at the end of A-track (mainly at U8–U9). Since typical RNAPIII terminators contain fewer than seven consecutive adenines, RNA release at U5–U7 (proximal terminator) likely represents the physiologically relevant mechanism. A set of elegant experiments with variant scaffolds revealed the unexpected role of the non-template DNA in termination. The A9-track appears to be sufficient to commit the complete and RNAPIIID to termination as RNA was released at U8–U9 even when the nontemplate strand was altered (Figure 1A). However, termination at the correct position is critical for reinitiation (Dieci et al., 2013), and release at U8–U9 may be irrelevant. The authors show that deletion of four amino acid residues in C37 or substitutions of the first four non-template thymidines abrogated pausing and RNA release at the ‘‘relevant’’ proximal terminator, whereas a substitution of the fifth thymidine retained pausing but abolished RNA release. These results provide a molecular explanation for earlier observations that C37/C53 were necessary for RNAPIII pausing within the terminator (Landrieux et al., 2006) and support a
974 Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc.
model in which sequence-specific interactions between C37 and the non-template DNA mediate the formation of a metastable pre-termination complex (PTC) (Figure 1B). In the PTC, several components collaborate to induce RNA release and enable productive reinitiation. The template strand A-track slows RNAPIII down and destabilizes the TEC by forming an unstable hybrid with the U-track in the nascent RNA. The non-template strand engages the C37/53 complex, additionally slowing RNAP. RNAPIII is prone to backtrack while transcribing through A-tracks, making it susceptible to C11, which rescues arrested TECs through cleavage of the nascent RNA. C37/53 and C11 (but not its RNA cleavage activity) are necessary for facilitated reinitiation. C37/53 and C11 may mediate conformational changes into a termination/reinitiation state—they form a complex located near the front edge of the moving RNAP and contain domains that approach the RNAPIII active site (Figure 1C). In addition, C37/53 interacts with the initiation factor TFIIIB, which remains bound to the promoter after initiation (Dieci et al., 2013). What are the structural changes that accompany the PTC formation? A T-track in the coding strand is part of the termination signal in bacteria (Gusarov and Nudler, 1999), archaea (Santangelo and Reeve, 2006), and RNAPIII, suggesting that termination could be mechanistically similar in these systems. In bacteria, the 9–10 bp RNA:DNA hybrid characteristic for an active TEC is thought to shorten to %7 bp upon invasion of the terminator RNA hairpin (Peters et al., 2011). We
Molecular Cell
Previews
Figure 1. Formation of the PTC (A) The T-track in the non-template strand and C37 are required for pausing and termination in the ‘‘correct,’’ proximal region, as shown by Arimbasseri and Maraia (2015). (B) The RNAP (gray oval) PTC is stabilized by interactions of the non-template DNA strand with C37 (orange) or by formation of the nascent RNA hairpin. (C) The proposed termination/reinitiation pathway. Following TFIIIB (green) and TFIIIC (not shown)-mediated recruitment, RNAPIII initiates at the start site (a bent arrow) and transcribes until it reaches the T-track. Domains of C11 (yellow), C37, and C53 (pink) extend toward the RNAP active site (a red dot). Interactions between the non-template T residues and C37 mediate pausing (at U4) and formation of a metastable PTC. Addition of subsequent UMP residues triggers further destabilization of the hybrid and RNA release. RNAP rebinds the promoter via interactions between C37 and TFIIIB, which remains associated with the promoter, whereas TFIIIC is dispensable for recycling on most genes. DNA wrapping could provide easy means for RNAP III handover. Alternatively, RNAPIII could slide back to the transcription start, only 100 nucleotides away on tRNA genes; however, the reported high occupancy of RNAPIII makes this scenario less likely (see Dieci et al., 2013 for a review). The conformation and occupancy of the transcribed DNA are yet unknown, as indicated by breaks therein.
propose that formation of the RNAPIII PTC also involves shortening of the RNA:DNA hybrid from the upstream side to 6–7 bp. Although in some species RNAPIII can terminate on as few as 4 Ts, 6 Ts appear to work in all cases. In a bacterial PTC, the shortened hybrid state is stabilized by the nascent RNA hairpin, whereas in RNAPIII PTC interactions of C37 with the non-template DNA may play a similar role (Figure 1B). It is noteworthy that the template DNA conformation in a bacterial initiation complex structure suggests that only a shorter 6–7 bp hybrid can form (Zuo and Steitz, 2015).
Thus, upon shortening of the RNA:DNA hybrid, the resulting PTC may resemble the initiation complex. The shortened U-rich hybrid would allow for RNA release from the enzyme, which could then rebind the promoter (Figure 1C). Isomerization into the PTC is likely mediated by largescale motions of the core subunits, such as ratcheting (Tagami et al., 2010), and the PTC may be additionally stabilized by RNAPIII initiation factors. Shortening of the RNA:DNA hybrid as the major transition accompanying the PTC formation provides an attractive explanation for the link between termination and reinitiation.
The sense or coding strand is commonly referred to as the non-template strand, an arguably disparaging term reflecting the fact that the coding strand is non-essential for transcription in vitro and is far less involved in RNA synthesis than the template strand. However, the non-template strand has long been known to play a leading role during initiation, where it is directly recognized by RNAP and accessory factors. Recent data indicate that interactions between the non-template DNA and the elongating RNAP (Vvedenskaya et al., 2014) or transcription factors (Yakhnin and Babitzke,
Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc. 975
Molecular Cell
Previews 2014) control RNA chain elongation. The report by Arimbasseri and Maraia provides compelling evidence for the importance of sequence-specific recognition of the non-template DNA during termination by yeast RNAPIII. Thus, besides encoding the primary sequences of noncoding RNAs and proteins indirectly, the non-template strand often carries the regulatory instructions that are recognized directly by diverse components of the transcription machinery during all stages of transcription.
REFERENCES
Santangelo, T.J., and Reeve, J.N. (2006). J. Mol. Biol. 355, 196–210.
Arimbasseri, A.G., and Maraia, R.J. (2015). Mol. Cell 58, this issue, 1124–1132.
Tagami, S., Sekine, S., Kumarevel, T., Hino, N., Murayama, Y., Kamegamori, S., Yamamoto, M., Sakamoto, K., and Yokoyama, S. (2010). Nature 468, 978–982.
Dieci, G., Bosio, M.C., Fermi, B., and Ferrari, R. (2013). Biochim. Biophys. Acta 1829, 331–341. Gusarov, I., and Nudler, E. (1999). Mol. Cell 3, 495–504. Landrieux, E., Alic, N., Ducrot, C., Acker, J., Riva, M., and Carles, C. (2006). EMBO J. 25, 118–128. Peters, J.M., Vangeloff, A.D., and Landick, R. (2011). J. Mol. Biol. 412, 793–813.
976 Molecular Cell 58, June 18, 2015 ª2015 Elsevier Inc.
Vvedenskaya, I.O., Vahedian-Movahed, H., Bird, J.G., Knoblauch, J.G., Goldman, S.R., Zhang, Y., Ebright, R.H., and Nickels, B.E. (2014). Science 344, 1285–1289. Yakhnin, A.V., and Babitzke, P. (2014). Curr. Opin. Microbiol. 18, 68–71. Zuo, Y., and Steitz, T.A. (2015). Mol. Cell 58, 534–540.