Transcript Cleavage by Thermus thermophilus RNA Polymerase
2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês
10.1074/jbc.m108737200
ISSN1083-351X
AutoresBrian P. Hogan, Thomas Hartsch, Dorothy A. Erie,
Tópico(s)Bacteriophages and microbial interactions
ResumoAll known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity ofThermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus andThermus aquaticus. The thermostable GreA factors contact the 3′-end of RNA, stimulate the intrinsic cleavage activity ofT. thermophilus RNA polymerase, and increase thekapp of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal “basic patch” region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase. All known multisubunit RNA polymerases possess the ability to endonucleolytically degrade the nascent RNA transcript. To gain further insight into the conformational changes that govern transcript cleavage, we have examined the effects of certain anions on the intrinsic transcript cleavage activity ofThermus thermophilus RNA polymerase. Our results indicate that the conformational transitions involved in transcript cleavage, and therefore backtracking, are anion-dependent. In addition to characterizing the intrinsic cleavage activity of T. thermophilus RNA polymerase, we have identified, cloned, and expressed a homolog of the prokaryotic transcript cleavage factor GreA from the extreme thermophiles, T. thermophilus andThermus aquaticus. The thermostable GreA factors contact the 3′-end of RNA, stimulate the intrinsic cleavage activity ofT. thermophilus RNA polymerase, and increase thekapp of the cleavage reaction 25-fold. In addition, we have identified a novel transcription factor in T. thermophilus and T. aquaticus that shares a high degree of sequence similarity with GreA, but has several residues that are not conserved with the N-terminal “basic patch” region of GreA. This protein, Gfh1, functions as an anti-GreA factor in vitro by reducing intrinsic cleavage and competing with GreA for a binding site on the polymerase. RNA polymerase potassium glutamate dithiothreitol ternary elongation complex RNA polymerase has been shown to possess a surprising activity that serves to release complexes from an arrested state. Specifically, RNA polymerase (RNAP)1appears to be able to catalyze the endo- and exonucleolytic cleavage of the RNA transcript, rapidly releasing the 3′-terminal fragment, which can be as large as 17 nucleotides in length, and resuming synthesis from the 5′-terminal fragment (1Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8899-8902Google Scholar, 2Surratt C.K. Milan S.C. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7983-7987Google Scholar). Two accessory proteins, GreA and GreB, have been found to stimulate this cleavage activity inEscherichia coli (1Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8899-8902Google Scholar, 3Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Google Scholar), and an ortholog of these factors (SII) has been found in eukaryotes (4Reines D. J. Biol. Chem. 1992; 267: 3795-3800Google Scholar, 5Sluder A.E. Greenleaf A.L. Price D.H. J. Biol. Chem. 1989; 264: 8963-8969Google Scholar). Transcript cleavage induced by GreA and GreB reduces abortive initiation (6Hsu L.M. Vo N.V. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11588-11592Google Scholar) and misincorporation (7Erie D.A. Hajiseyedjavadi O. Young M.C. von Hippel P.H. Science. 1993; 262: 867-873Google Scholar) and regulates pausing and arrest during elongation (3Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Google Scholar, 7Erie D.A. Hajiseyedjavadi O. Young M.C. von Hippel P.H. Science. 1993; 262: 867-873Google Scholar, 8Artsimovitch I. Landick R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7090-7095Google Scholar, 9Marr M.T. Roberts J.W. Mol. Cell. 2000; 6: 1275-1285Google Scholar). Taken together, these results suggest that transcript cleavage plays an important role in maintaining processive and accurate synthesis of the RNA transcript in vivo. There is strong evidence that this cleavage activity resides on RNA polymerase itself (10Rudd M.D. Izban M.G. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8057-8061Google Scholar, 11Wang D. Hawley D.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 843-847Google Scholar). In addition, it has been suggested that the same amino acids that are responsible for nucleotide incorporation may be responsible for this cleavage activity (10Rudd M.D. Izban M.G. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8057-8061Google Scholar, 11Wang D. Hawley D.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 843-847Google Scholar). If this hypothesis is true, it would indicate large scale movements of the catalytic site relative to the RNA transcript and the DNA template (12Mustaev A. Kashlev M. Zaychikov E. Grachev M. Goldfarb A. J. Biol. Chem. 1993; 268: 19185-19187Google Scholar, 13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar). Such conformational changes have been investigated in vitro using stalled complexes formed by NTP deprivation or physical obstruction (15Arndt K.M. Chamberlin M.J. J. Mol. Biol. 1990; 213: 79-108Google Scholar, 16Krummel B. Chamberlin M.J. J. Mol. Biol. 1992; 225: 221-237Google Scholar, 17Xue Y. Hogan B.P. Erie D.A. Biochemistry. 2000; 39: 14356-14362Google Scholar). Studies of these complexes suggest that the formation of a reverse-translocated, or backtracked state, is a necessary step in the transcript cleavage reaction (13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar). Backtracking repositions the catalytic site residues of RNAP upstream on the RNA and DNA (13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar,16Krummel B. Chamberlin M.J. J. Mol. Biol. 1992; 225: 221-237Google Scholar, 18Reeder T.C. Hawley D.K. Cell. 1996; 87: 767-777Google Scholar) while displacing the 3′-end of the RNA transcript from the catalytic site. It has been suggested that the 3′-end of the RNA is extruded through the secondary channel of RNAP in a backtracked complex (8Artsimovitch I. Landick R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7090-7095Google Scholar, 19Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Google Scholar), which presumably positions the internal phosphodiester bonds of the transcript for hydrolysis by RNAP.For backtracking and transcript cleavage to occur, interactions between RNAP and upstream and downstream DNA need to be altered, as well as single-stranded DNA-RNAP, RNA-RNAP, and DNA-RNA hybrid interactions. Given the number of protein-nucleic acid interactions within the ternary elongation complex, it is likely that the conformational changes associated with backtracking and transcript cleavage may be sensitive to salt concentration (20Leirmo S. Harrison C. Cayley D.S. Burgess R.R. Record Jr., M.T. Biochemistry. 1987; 26: 2095-2101Google Scholar). Different components of the transcription cycle that involve large conformational changes in RNAP and nucleic acid are indeed sensitive to certain electrolytes. For example, RNAP can form an open complex at the λPR promoter in 200 mm potassium glutamate (Kglu), but not in buffer containing 200 mm potassium chloride (KCl) (20Leirmo S. Harrison C. Cayley D.S. Burgess R.R. Record Jr., M.T. Biochemistry. 1987; 26: 2095-2101Google Scholar). Similarly, individual anions within the Hofmeister series can either increase or decrease the rate of elongation and have quantitative effects on pause half-lives (21Chan C.L. Landick R. J. Mol. Biol. 1997; 268: 37-53Google Scholar).In this report, we present the characterization of the intrinsic cleavage activity of Thermus thermophilus RNAP. We find that the intrinsic cleavage activity of the polymerase is significantly influenced by the anion concentration of the transcription buffer. In addition to examining factor-independent transcript cleavage, we have also characterized the effects of two GreA homologs on the cleavage activity of T. thermophilus RNAP. One of the two Gre-like factors is a true homolog of GreA, containing the highly conserved “basic patch” region within its N-terminal region (22Kulish D. Lee J. Lomakin I. Nowicka B. Das A. Darst S. Normet K. Borukhov S. J. Biol. Chem. 2000; 275: 12789-12798Google Scholar), and possessing transcript cleavage properties similar to E. coliGreA. Interestingly, the second Gre-like factor shares a high degree of sequence identity with GreA in its C-terminal region, but several residues within its N-terminal region differ from those in the N-terminal basic patch region of GreA. This factor is only found in a limited set of organisms and appears to function as an anti-GreA factor in vitro. Because of its high degree of sequence similarity but lack of functional homology to GreA, we have named this new factor Gfh1 for Gre FactorHomolog 1. RNA polymerase has been shown to possess a surprising activity that serves to release complexes from an arrested state. Specifically, RNA polymerase (RNAP)1appears to be able to catalyze the endo- and exonucleolytic cleavage of the RNA transcript, rapidly releasing the 3′-terminal fragment, which can be as large as 17 nucleotides in length, and resuming synthesis from the 5′-terminal fragment (1Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8899-8902Google Scholar, 2Surratt C.K. Milan S.C. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7983-7987Google Scholar). Two accessory proteins, GreA and GreB, have been found to stimulate this cleavage activity inEscherichia coli (1Borukhov S. Polyakov A. Nikiforov V. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8899-8902Google Scholar, 3Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Google Scholar), and an ortholog of these factors (SII) has been found in eukaryotes (4Reines D. J. Biol. Chem. 1992; 267: 3795-3800Google Scholar, 5Sluder A.E. Greenleaf A.L. Price D.H. J. Biol. Chem. 1989; 264: 8963-8969Google Scholar). Transcript cleavage induced by GreA and GreB reduces abortive initiation (6Hsu L.M. Vo N.V. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11588-11592Google Scholar) and misincorporation (7Erie D.A. Hajiseyedjavadi O. Young M.C. von Hippel P.H. Science. 1993; 262: 867-873Google Scholar) and regulates pausing and arrest during elongation (3Borukhov S. Sagitov V. Goldfarb A. Cell. 1993; 72: 459-466Google Scholar, 7Erie D.A. Hajiseyedjavadi O. Young M.C. von Hippel P.H. Science. 1993; 262: 867-873Google Scholar, 8Artsimovitch I. Landick R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7090-7095Google Scholar, 9Marr M.T. Roberts J.W. Mol. Cell. 2000; 6: 1275-1285Google Scholar). Taken together, these results suggest that transcript cleavage plays an important role in maintaining processive and accurate synthesis of the RNA transcript in vivo. There is strong evidence that this cleavage activity resides on RNA polymerase itself (10Rudd M.D. Izban M.G. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8057-8061Google Scholar, 11Wang D. Hawley D.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 843-847Google Scholar). In addition, it has been suggested that the same amino acids that are responsible for nucleotide incorporation may be responsible for this cleavage activity (10Rudd M.D. Izban M.G. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8057-8061Google Scholar, 11Wang D. Hawley D.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 843-847Google Scholar). If this hypothesis is true, it would indicate large scale movements of the catalytic site relative to the RNA transcript and the DNA template (12Mustaev A. Kashlev M. Zaychikov E. Grachev M. Goldfarb A. J. Biol. Chem. 1993; 268: 19185-19187Google Scholar, 13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar). Such conformational changes have been investigated in vitro using stalled complexes formed by NTP deprivation or physical obstruction (15Arndt K.M. Chamberlin M.J. J. Mol. Biol. 1990; 213: 79-108Google Scholar, 16Krummel B. Chamberlin M.J. J. Mol. Biol. 1992; 225: 221-237Google Scholar, 17Xue Y. Hogan B.P. Erie D.A. Biochemistry. 2000; 39: 14356-14362Google Scholar). Studies of these complexes suggest that the formation of a reverse-translocated, or backtracked state, is a necessary step in the transcript cleavage reaction (13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar). Backtracking repositions the catalytic site residues of RNAP upstream on the RNA and DNA (13Komissarova N. Kashlev M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1755-1760Google Scholar, 14Nudler E. Mustaev A. Lukhtanov E. Goldfarb A. Cell. 1997; 89: 33-41Google Scholar,16Krummel B. Chamberlin M.J. J. Mol. Biol. 1992; 225: 221-237Google Scholar, 18Reeder T.C. Hawley D.K. Cell. 1996; 87: 767-777Google Scholar) while displacing the 3′-end of the RNA transcript from the catalytic site. It has been suggested that the 3′-end of the RNA is extruded through the secondary channel of RNAP in a backtracked complex (8Artsimovitch I. Landick R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7090-7095Google Scholar, 19Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Google Scholar), which presumably positions the internal phosphodiester bonds of the transcript for hydrolysis by RNAP. For backtracking and transcript cleavage to occur, interactions between RNAP and upstream and downstream DNA need to be altered, as well as single-stranded DNA-RNAP, RNA-RNAP, and DNA-RNA hybrid interactions. Given the number of protein-nucleic acid interactions within the ternary elongation complex, it is likely that the conformational changes associated with backtracking and transcript cleavage may be sensitive to salt concentration (20Leirmo S. Harrison C. Cayley D.S. Burgess R.R. Record Jr., M.T. Biochemistry. 1987; 26: 2095-2101Google Scholar). Different components of the transcription cycle that involve large conformational changes in RNAP and nucleic acid are indeed sensitive to certain electrolytes. For example, RNAP can form an open complex at the λPR promoter in 200 mm potassium glutamate (Kglu), but not in buffer containing 200 mm potassium chloride (KCl) (20Leirmo S. Harrison C. Cayley D.S. Burgess R.R. Record Jr., M.T. Biochemistry. 1987; 26: 2095-2101Google Scholar). Similarly, individual anions within the Hofmeister series can either increase or decrease the rate of elongation and have quantitative effects on pause half-lives (21Chan C.L. Landick R. J. Mol. Biol. 1997; 268: 37-53Google Scholar). In this report, we present the characterization of the intrinsic cleavage activity of Thermus thermophilus RNAP. We find that the intrinsic cleavage activity of the polymerase is significantly influenced by the anion concentration of the transcription buffer. In addition to examining factor-independent transcript cleavage, we have also characterized the effects of two GreA homologs on the cleavage activity of T. thermophilus RNAP. One of the two Gre-like factors is a true homolog of GreA, containing the highly conserved “basic patch” region within its N-terminal region (22Kulish D. Lee J. Lomakin I. Nowicka B. Das A. Darst S. Normet K. Borukhov S. J. Biol. Chem. 2000; 275: 12789-12798Google Scholar), and possessing transcript cleavage properties similar to E. coliGreA. Interestingly, the second Gre-like factor shares a high degree of sequence identity with GreA in its C-terminal region, but several residues within its N-terminal region differ from those in the N-terminal basic patch region of GreA. This factor is only found in a limited set of organisms and appears to function as an anti-GreA factor in vitro. Because of its high degree of sequence similarity but lack of functional homology to GreA, we have named this new factor Gfh1 for Gre FactorHomolog 1. We thank S. Darst for the gift of T. aquaticus cells, L. Spremulli, S. Holmes, S. Holtschlag, and Y. Xue for technical assistance, and K. Hogan for help in preparing the manuscript.
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