Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4·Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae
2007; Elsevier BV; Volume: 283; Issue: 7 Linguagem: Inglês
10.1074/jbc.m708746200
ISSN1083-351X
AutoresVinaya Sampath, Bindu Balakrishnan, Jiyoti Verma‐Gaur, Silvia Onesti, Parag P. Sadhale,
Tópico(s)Fungal and yeast genetics research
ResumoTwo subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4·Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase. Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4·Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase. Studies of transcriptional regulation have focused mainly on the role of DNA-bound regulatory proteins and their contacts with the general transcription factors, mediator, and other accessory proteins in the transcriptional machinery. In most eukaryotes, the 12-subunit RNA polymerase II (Pol II) 4The abbreviations used are: Pol IIRNA polymerase IIRNPribonucleoproteinOBoligonucleotide/oligosaccharide bindingYPDyeast extract peptone dextrose mediumTAPtandem affinity purificationWTwild typeaaamino acids. is thought to have little or no influence on the regulation of transcription. Rpb4 and Rpb7 form a subcomplex within the polymerase that dissociates easily under mild denaturing or nondenaturing conditions and shows variable association with the polymerase at different growth stages, leading to the suggestion that the subcomplex could be analogous to the σ subunit of the bacterial RNA polymerase (1Choder M. Young R.A. Mol. Cell. Biol. 1993; 13: 6984-6991Crossref PubMed Scopus (115) Google Scholar, 2Edwards A.M. Kane C.M. Young R.A. Kornberg R.D. J. Biol. Chem. 1991; 266: 71-75Abstract Full Text PDF PubMed Google Scholar, 3Kolodziej P.A. Woychik N. Liao S.M. Young R.A. Mol. Cell. Biol. 1990; 10: 1915-1920Crossref PubMed Scopus (100) Google Scholar). Whether such a regulatory role can be ascribed to the subcomplex has been a matter of some debate, but the phenotypes of the deletion mutants and the interactions mediated by these subunits suggest that they might have some regulatory role in stress response and transcription (4Sampath V. Sadhale P. IUBMB Life. 2005; 57: 93-102Crossref PubMed Scopus (31) Google Scholar). RNA polymerase II ribonucleoprotein oligonucleotide/oligosaccharide binding yeast extract peptone dextrose medium tandem affinity purification wild type amino acids. Rpb7 is essential for survival of Saccharomyces cerevisiae, whereas Rpb4 is not (5McKune K. Richards K.L. Edwards A.M. Young R.A. Woychik N.A. Yeast. 1993; 9: 295-299Crossref PubMed Scopus (76) Google Scholar). However, rpb4Δ strains are temperature-sensitive and cold-sensitive, show poor recovery from stationary phase, are defective in sporulation (a response to severe nutritional starvation), and are predisposed to pseudohyphae formation (a response to mild nutritional starvation) (4Sampath V. Sadhale P. IUBMB Life. 2005; 57: 93-102Crossref PubMed Scopus (31) Google Scholar). Apart from its roles in stress response, Rpb4 is involved in transcription under moderate and extreme temperatures (6Pillai B. Verma J. Abraham A. Francis P. Kumar Y. Tatu U. Brahmachari S.K. Sadhale P.P. J. Biol. Chem. 2003; 278: 3339-3346Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The polymerase lacking Rpb4 and Rpb7 is defective for promoter-dependent initiation of transcription but not for promoter-independent chain elongation (2Edwards A.M. Kane C.M. Young R.A. Kornberg R.D. J. Biol. Chem. 1991; 266: 71-75Abstract Full Text PDF PubMed Google Scholar). Pol II activity at extreme temperatures is dependent on the presence of Rpb4, leading to a suggestion that the Rpb4·Rbp7 subcomplex controls the stability of Pol II under extreme temperatures (7Rosenheck S. Choder M. J. Bacteriol. 1998; 180: 6187-6192Crossref PubMed Google Scholar). However, rpb4Δ strain is also defective for activated transcription from a subset of genes at moderate temperatures (8Pillai B. Sampath V. Sharma N. Sadhale P. J. Biol. Chem. 2001; 276: 30641-30647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Early studies showed that Pol II purified from rpb4Δ strain lacks Rpb7, suggesting that the Rpb4 stabilizes the interaction of Rpb7 with the rest of the Pol II (9Woychik N.A. Young R.A. Mol. Cell. Biol. 1989; 9: 2854-2859Crossref PubMed Scopus (149) Google Scholar, 10Sheffer A. Varon M. Choder M. Mol. Cell. Biol. 1999; 19: 2672-2680Crossref PubMed Scopus (57) Google Scholar). Consistent with such a hypothesis, overexpression of Rpb7 can partially rescue the temperature sensitivity and sporulation defects of the rpb4Δ strain (10Sheffer A. Varon M. Choder M. Mol. Cell. Biol. 1999; 19: 2672-2680Crossref PubMed Scopus (57) Google Scholar, 11Sharma N. Sadhale P.P. J. Genet. 1999; 78: 149-156Crossref Scopus (12) Google Scholar). In addition, overexpression of RPB7 can rescue activated transcription only from certain stress promoters but not from the nonstress promoters, suggesting that ScRpb7 may not have a generalized role in promoter-dependent transcription in vivo (8Pillai B. Sampath V. Sharma N. Sadhale P. J. Biol. Chem. 2001; 276: 30641-30647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Rpb4 plays a Rpb7-independent role in promoting mRNA export and maintaining transcript stability of a subset of genes (12Lotan R. Bar-On V.G. Harel-Sharvit L. Duek L. Melamed D. Choder M. Genes Dev. 2005; 19: 3004-3016Crossref PubMed Scopus (102) Google Scholar, 13Farago M. Nahari T. Hammel C. Cole C.N. Choder M. Mol. Biol. Cell. 2003; 14: 2744-2755Crossref PubMed Scopus (54) Google Scholar). On the other hand, overexpression of Rpb7 in rpb4Δ and the Σ1278b (a genetic background that shows predisposition to forming pseudohyphae) strains can lead to exaggeration of pseudohyphal growth (14Khazak V. Sadhale P.P. Woychik N.A. Brent R. Golemis E.A. Mol. Biol. Cell. 1995; 6: 759-775Crossref PubMed Scopus (67) Google Scholar, 15Singh S.R. Rekha N. Pillai B. Singh V. Naorem A. Sampath V. Srinivasan N. Sadhale P.P. Nucleic Acids Res. 2004; 32 (Print 2004): 201-210Crossref PubMed Scopus (8) Google Scholar, 16Singh S.R. Pillai B. Balakrishnan B. Naorem A. Sadhale P.P. Biochem. Biophys. Res. Commun. 2007; 356: 266-272Crossref PubMed Scopus (10) Google Scholar). Recently, it has been shown that Rpb4 and Rpb7 may also exist as a subcomplex outside the context of Pol II (17Selitrennik M. Duek L. Lotan R. Choder M. Eukaryot. Cell. 2006; 5: 2092-2103Crossref PubMed Scopus (41) Google Scholar). A single model that will explain all the phenotypes observed with the mutants of the two subunits is yet to be proposed (4Sampath V. Sadhale P. IUBMB Life. 2005; 57: 93-102Crossref PubMed Scopus (31) Google Scholar). Rpb7 is highly conserved from archaea to humans with sequence identity at 40–70% between the eukaryotic homologs. Sequence alignment of the eukaryotic Rpb7 subunits shows that the central region (residues 63–82) essential for viability of S. cerevisiae is highly conserved (18Sadhale P.P. Woychik N.A. Mol. Cell. Biol. 1994; 14: 6164-6170Crossref PubMed Scopus (44) Google Scholar). The high sequence conservation of Rpb7 is mirrored by the ability of many of the eukaryotic homologs (from humans, Schizosaccharomyces pombe, Candida albicans, and Dictyostelium discoideum) to rescue the lethality of S. cerevisiae rpb7Δ strain (14Khazak V. Sadhale P.P. Woychik N.A. Brent R. Golemis E.A. Mol. Biol. Cell. 1995; 6: 759-775Crossref PubMed Scopus (67) Google Scholar, 15Singh S.R. Rekha N. Pillai B. Singh V. Naorem A. Sampath V. Srinivasan N. Sadhale P.P. Nucleic Acids Res. 2004; 32 (Print 2004): 201-210Crossref PubMed Scopus (8) Google Scholar, 19Shpakovski G.V. Gadal O. Labarre-Mariotte S. Lebedenko E.N. Miklos I. Sakurai H. Proshkin S.A. Van Mullem V. Ishihama A. Thuriaux P. J. Mol. Biol. 2000; 295: 1119-1127Crossref PubMed Scopus (26) Google Scholar). The three-dimensional structure determination of ScRpb4·Rbp7 and their homologs from humans and archaea (RpoF/RpoE) show that the major features of the structures are superimposable to a large extent (20Armache K.J. Mitterweger S. Meinhart A. Cramer P. J. Biol. Chem. 2005; 280: 7131-7134Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 21Meka H. Werner F. Cordell S.C. Onesti S. Brick P. Nucleic Acids Res. 2005; 33: 6435-6444Crossref PubMed Scopus (56) Google Scholar, 22Todone F. Brick P. Werner F. Weinzierl R.O. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Rpb7 consists of two putative single-stranded RNA-binding domains; the N terminus folds into a truncated RNP structure, whereas the C-terminal half of the molecule forms an OB fold. The bottom region of the RNP domain (the “tip loop”) comprises many surface-exposed residues that are highly conserved, and the crystal structure of the 12-subunit complex shows that this region is the main anchorage point for the interaction with the Pol II core (20Armache K.J. Mitterweger S. Meinhart A. Cramer P. J. Biol. Chem. 2005; 280: 7131-7134Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Despite the structural similarity to other RNP domains, site-directed mutagenesis experiments suggest that the residues contributing to RNA binding in the N-terminal domain are not located on the canonical face of the RNP fold (21Meka H. Werner F. Cordell S.C. Onesti S. Brick P. Nucleic Acids Res. 2005; 33: 6435-6444Crossref PubMed Scopus (56) Google Scholar). The presence of an OB fold in the C-terminal domain was predicted based on the presence of a sequence pattern characteristic of the S1 motif (a subgroup of OB fold proteins) (23Orlicky S.M. Tran P.T. Sayre M.H. Edwards A.M. J. Biol. Chem. 2001; 276: 10097-10102Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The OB fold comprises a five-stranded anti-parallel β-barrel with a number of conserved surface-exposed residues on one face of the barrel, which have been shown to be involved in RNA binding (21Meka H. Werner F. Cordell S.C. Onesti S. Brick P. Nucleic Acids Res. 2005; 33: 6435-6444Crossref PubMed Scopus (56) Google Scholar). A unique feature of the Rpb7 OB fold is the presence of a long insertion between strand B3 and B4, where a short 310 helix (which is present in all the S1 domains) is followed by a three-stranded anti-parallel β-sheet. The crystal structure of the archaeal complex suggested a model for the function of the heterodimer in which the S1 motif of Rpb7 interacts with the nascent RNA transcript, possibly assisted by the truncated RNP domain (22Todone F. Brick P. Werner F. Weinzierl R.O. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The location of the Rpb4·Rbp7 heterodimer in the polymerase based on the low resolution crystal structure of the 12-subunit Pol II (24Armache K.J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, 25Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6969-6973Crossref PubMed Scopus (222) Google Scholar) confirmed that Rpb7 is in a position to interact with the nascent RNA transcript released from the transcribing complex. This prediction has been recently validated by UV cross-linking studies (26Ujvari A. Luse D.S. Nat. Struct. Mol. Biol. 2006; 13: 49-54Crossref PubMed Scopus (83) Google Scholar). Although the subcomplex has been shown to bind the nascent transcript, a clearly defined molecular function for the subcomplex has not yet emerged. The structure of the 12-subunit RNA polymerase from S. cerevisiae determined at higher resolution allowed a more detailed view of the interaction between the Rpb4·Rpb7 complex and the polymerase core (20Armache K.J. Mitterweger S. Meinhart A. Cramer P. J. Biol. Chem. 2005; 280: 7131-7134Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The tip loop of the RNP domain of Rpb7 interacts with a region of Pol II made up of the linker region of Rpb1 and Rpb6. Furthermore, the authors propose that the N-terminal residues of Rpb4 maybe involved in the interaction with Rpb2. However, the relative contributions of the reported points of interaction between the subcomplex and the core polymerase II and the consequences of disrupting them have not been studied in detail. Given the essential nature of Rpb7, the complexity of its roles in multiple phenotypes and the lack of useful information from deletion-based analyses, we decided to use genetic screens to isolate mutants of RPB7. In this report, we present the characterization of a loss of function mutant rpb7–7, which, in combination with a previously reported deletion of Rpb4 (27Sampath V. Rekha N. Srinivasan N. Sadhale P. J. Biol. Chem. 2003; 278: 51566-51576Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), reveals how the subcomplex interacts with the rest of the Pol II. Yeast Strains–The yeast strains used in this study are listed in Table 1. These strains were transformed with the appropriate plasmids and assayed for various phenotypes. Yeast transformations were performed routinely using the modified lithium acetate protocol that does not involve heat treatment of cells (28Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1983Google Scholar). For high efficiency transformation for the genetic screens, the method of Finley and Brent (29Finley R.L. Brent R. Bartel P.L. Fields S. The yeast Two-hybrid System. Oxford University Press, Oxford, UK1997: 197-214Google Scholar) was followed. Yeast strains were grown in rich YPD medium or in synthetic dextrose medium containing 2% glucose or 2% galactose as a carbon source and the required amino acids.TABLE 1List of strains used in this workStrainGenotypeSourceSY10MATa, leu2-3, lys2-112, ura3-52, rpb4Δ::HIS3Ref. 8Pillai B. Sampath V. Sharma N. Sadhale P. J. Biol. Chem. 2001; 276: 30641-30647Abstract Full Text Full Text PDF PubMed Scopus (33) Google ScholarSYD1011MATa/α, his3Δ200/his3Δ200, ura3-52/ura3-52, leu2-3,/leu2-3,112, lys2-1/lys2-1, rpb4Δ::HIS3/rpb4Δ::HIS3Ref. 15Singh S.R. Rekha N. Pillai B. Singh V. Naorem A. Sampath V. Srinivasan N. Sadhale P.P. Nucleic Acids Res. 2004; 32 (Print 2004): 201-210Crossref PubMed Scopus (8) Google ScholarSYD7MATa/α, ADE2/ade2-101, his3-Δ200/his3-Δ200, leu2-3/leu2-3, TRP1/trp1-901, lys2-112/lys2-112, RPB7/rpb7Δ::LEU2Ref. 15Singh S.R. Rekha N. Pillai B. Singh V. Naorem A. Sampath V. Srinivasan N. Sadhale P.P. Nucleic Acids Res. 2004; 32 (Print 2004): 201-210Crossref PubMed Scopus (8) Google ScholarBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Euroscarf, GermanyY07005MATa rpb4Δ::kanr his3Δ1 leu2Δ0 met15Δ0 ura3Δ0This studySC1126MATa; ade2; arg4; leu2-3,112; trp1-289; ura3-52; YIL021W::TAP-K.I.URA3Euroscarf, GermanyYKL200MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, can1-100, UBR1::GAL-HA-UBR1(HIS3)Euroscarf, GermanyN-deg 7MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, can1-100, UBR1::GAL-HA-UBR1(HIS3), N-deg Rpb7(KANr)This study Open table in a new tab Plasmids–The gapped plasmid (containing sequences 650 bp upstream and downstream of Rpb7 open reading frame separated by a HindIII site) pVS134 used in the genetic screen to isolate loss of function mutations in Rpb7 was generated by subcloning the 1.5-kb PvuII fragment from pBP86 in between the PvuII sites of pPS7 (YEplac195). The plasmid pVS154 expressing the rpb7–7 mutant and the plasmid pSP186 expressing wild type RPB7 from pPS7 were used to separate the N- and the C-terminal mutations of rpb7–7. The plasmid, pVS357 expressing the rpb7–7 N-terminal mutations was generated by a three-way ligation between a 0.9-kb EcoRI-DraI fragment from pVS154, a 0.5-kb DraI-SalI fragment from pSP186, and the EcoRI-SalI-digested pPS7. The plasmid pVS358 expressing the rpb7–7 C-terminal mutations was generated by a three-way ligation between a 0.95-kb BamHI-DraI fragment from pVS154, a 0.9-kb DraI-SalI fragment from pSP186, and the BamHI-SalI-digested pPS7. Plasmids pBB651 and pBB652 are the WT RPB7 and the rpb7–7 mutant alleles from the pVS186 and pVS154 plasmid, respectively, subcloned in the plasmid pPS6 (YEplac112) with TRP1 auxotrophic marker to enable us to accommodate all the plasmids used in this work. Plasmid YKL 187 for amplification of the degron cassette was obtained from Euroscarf (Frankfurt, Germany). Construction of the TAP-tagged Strains–The N-Deg7 strain was generated by fusing the Rpb7 open reading frame in YKL200 strain with the N-degron cassette amplified with the plasmid YKL187 as template and primers (5′-GTT TCT CCT CCT ACA CCA TTC TCC TTT GCG ATT AGA CAG TGG GAA AGT CCA TTA AGG CGC GCC AGA TCT G-3′ and 5′-AAG GAC GGA TGA AGG GTA ATA TTA AGC GAA AGG TCT TTA ATA AAA AAC ATG GCA CCC GCT CCA GCG CCT G-3′) (30Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar). Strain SC1126 was crossed with the Y07005 (rpb4Δ) strain to obtain rpb4Δ strain containing the TAP-tagged Rpb3 fusion. This haploid transformed with the different RPB4 alleles was crossed to the N-Deg7 strain transformed with different RPB7 alleles and segregants expressing C-terminal TAP tag Rpb3 and N-deg-Rpb7 fusion proteins in both wild type as well as rpb4Δ background were obtained. The haploids were selected on YPDCuSO4 with 500 μg/ml G418 because the N-deg Rpb7 open reading frame is under the control of CUP1 promoter, and the rpb4Δ is marked with kanr resistance. Colony size, rate of growth, and temperature sensitivity were used as parameters for initial screening of rpb4Δ strains and wild type strain because rpb4Δ strains are temperature-sensitive and slow growing with small colony size. The haploid isolates were further confirmed by PCR specific for the N-Deg Rpb7 module and the rpb4Δ locus and by Western blotting with New Zealand White rabbit serum to detect the TAP-tagged moiety in WT and rpb4Δ strains. To check survival of the cells at 37 °C in the presence of high levels of the galactose-inducible Ubr1 protein, both the strains were grown on yeast extract peptone galactose medium and incubated at 37 °C. Temperature Sensitivity, Sporulation, and Pseudohyphal Growth–These assays were performed essentially as described before (8Pillai B. Sampath V. Sharma N. Sadhale P. J. Biol. Chem. 2001; 276: 30641-30647Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The assay for temperature sensitivity was performed on synthetic dextrose plates containing 2% glucose at 25 or 37 °C. The assays for sporulation and pseudohyphal growth were done on 1% potassium acetate plates and on synthetic low ammonia dextrose medium, respectively. Random Spore Analysis–Sporulated cultures were treated with lyticase (1 mg/ml in 1 m sorbitol) for 30 min to 1 h, vortexed with an equal volume of mineral oil for 2 min, and centrifuged for 30 s. The mineral oil layer enriched with hydrophobic spores was plated on appropriate selection medium. TAP Tag-based Pol II Pulldowns (31Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3998) Google Scholar)–The strains with C-terminally TAP-tagged RPB3 in rpb4Δ genetic background carrying either wild type or Rpb4-(33–221) mutant expressing plasmids pNS114 and pVS378 and with rpb7-7 expressing plasmid pVS154 were grown overnight in YPDCuSO4 at 26 °C and diluted to a density of 5 × 106 cells/ml in 50 ml of fresh YPDCuSO4 medium. The cells were pelleted and washed in sterile water, freeze-thawed in liquid nitrogen, and then resuspended in 250 μl of whole cell extract lysis buffer (20 mm HEPES, pH 7.9, 10% glycerol, 0.5 mm EDTA, 300 mm potassium acetate, 2 mm dithiothreitol, and 0.05% Nonidet P-40 with protease inhibitors). The cells were lysed with the addition of chilled glass beads by four cycles of 1 min of vortexing alternating with 5 min cooling on ice. The lysates were clarified at 13,000 rpm for 20 min at 4 °C. The protein concentrations were determined by Bradford assay (Invitrogen). Approximately 1.5 mg of total protein was used for TAP tag purification or immunoprecipitation. TAP-tagged extracts were incubated with 40 μl of rabbit IgG-agarose at 4 °C for 1 h. The beads, equilibrated in TEV protease cleavage buffer, were washed three times with 1 ml of the same buffer. The proteins were eluted from the beads with the addition of 2.5 units of TEV protease in 50 μl of TEV protease cleavage buffer. The eluted proteins were analyzed by Western blotting to identify the interaction of the subcomplex with the rest of the Pol II. The N-deg Rpb7 and rpb7–7 alleles were monitored using α-Rpb7 antibodies generated in the laboratory. The α-Rpb4 antibody procured from Neoclone Inc. was raised against the N-terminal region of Rpb4 and hence would not recognize the Rpb4-(33–221) allele described here. Although the Rpb4-(33–221) allele cannot be recognized by the α-Rpb4 antibody, the protein is being made and is stable because it can complement the stress response defects of rpb4Δ (27Sampath V. Rekha N. Srinivasan N. Sadhale P. J. Biol. Chem. 2003; 278: 51566-51576Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Genetic Screen Allowed Isolation of Conditional Mutants of RPB7–We used a genetic screen to isolate conditional “loss of function” alleles of RPB7. Fig. 1A shows a schematic representation of the screen used to isolate mutants of Rpb7 gene carried on a 2μ plasmid. Briefly, a gapped plasmid was generated that had 650 bp of the 5′- and 3′-untranslated regions of RPB7 at its linearized ends. This gapped plasmid was transformed along with a PCR-mutagenized RPB7 gene pool into the heterozygous RPB7/rpb7Δ strain, SYD7. At the time of carrying out the screen, the only available rpb7Δ mutant was marked with LEU2, and the complementation studies were carried out with the RPB7 gene on a plasmid carrying the URA3 gene. Hence a conventional plasmid shuffle screen was not possible. The transformants were pooled and sporulated, and haploids were generated by random spore analysis. rpb7Δ haploids carrying the in vivo recombined plasmid were selected based on their abilities to grow in the absence of leucine and uracil, ensuring the selection of rpb7Δ cells and the plasmid, respectively. The random spore analysis was standardized to ensure minimal contamination with diploids, and the haploids were confirmed by mating type PCR analysis. These haploids were further screened for temperature sensitivity at 37 °C. The plasmids isolated from the putative mutants were analyzed for various stress phenotypes associated with rpb4Δ and rpb7Δ strains. Of the 6000 transformants screened, 70 haploids showed the temperature-sensitive phenotype. Of these, six alleles that showed consistent phenotypes after multiple rounds of screening were used for further characterization. These mutants were compared with wild type RPB7 in various assays in rpb7Δ and rpb4Δ strains. The ability of the plasmid borne alleles to complement the lethality of rpb7Δ strain was tested by retransforming the RPB7/rpb7Δ (SYD7) and carrying out random spore analysis. We defined the percentage of survival as the percentage of haploids carrying a certain genotype among all haploids generated from the sporulated SYD7 strain by random spore analysis. Complementation of the lethality of rpb7Δ by wild type RPB7 results in 50% survival. All of the mutants analyzed showed ability to complement rpb7Δ at room temperature (data not shown), but the sensitivity of the mutants to high temperature varied to a great extent (Fig. 1B). As mentioned earlier and also seen in Fig. 2, rpb4Δ mutant is temperature-sensitive, defective in sporulation, and predisposed to pseudohyphae formation. Overexpression of RPB7 in rpb4Δ rescues the temperature sensitivity at 34 °C but not at 37 °C. It also rescues the sporulation defect but exaggerates the pseudohyphal morphology. The six mutants analyzed for the rescue of the above phenotypes in rpb4Δ strain showed that except for rpb7–7, all alleles were able to rescue temperature sensitivity, whereas some alleles along with rpb7–7 were unable to rescue sporulation defect. Interestingly, all of them exaggerated the pseudohyphal phenotype. Table 2 summarizes the phenotypes of these mutants.TABLE 2Summary of phenotypes of the different loss of function mutants isolated in the genetic screen in rpb7Δ and rpb4Δ strainsAlleleSequence changesrpb7Δrpb4ΔGrowth at 38 °CProtein levelGrowth at 34 °CSporulationPseudohyphal growthProtein levelRPB7None+++++++rpb7-7F70L, V72M, A123E, S125T++--+++rpb7-18D50A, T138P--+±++-rpb7-34I147G+++++++++rpb7-61S162T±++-+++rpb7-62S162T-+++++++rpb7-63ND±++-+++ Open table in a new tab In this report, we present detailed characterization of the rpb7–7 mutant, which has provided us with a new insight into the interaction between Rpb7 and Rpb4 and the function of the subcomplex. The rpb7–7 mutant was chosen because it has interesting phenotypes in the absence of Rpb4 but behaved like the WT protein in the presence of Rpb4. The plasmid from rpb7–7 mutant was isolated and retransformed into SYD7 to confirm that the lethality of rpb7Δ can be complemented by the mutant as well as RPB7. In addition, the mutant allele does not compromise the growth of rpb7Δ significantly at higher temperature (Fig. 1B). Interestingly, the rpb7–7 allele, when compared with the wild type RPB7 expressed from a similar 2μ plasmid, shows a clear defect in its ability to rescue the temperature sensitivity and sporulation defects of rpb4Δ strain (Fig. 2, A and B). However, its ability to enhance the pseudohyphal morphology of rpb4Δ strain is not affected (Fig. 2C). The steady state protein level of the rpb7–7 allele in rpb4Δ and rpb7Δ strains is stable and expressed at similar levels as wild type Rpb7 in both rpb4Δ and rpb7Δ strains (data not shown). Thus the defective sporulation and temperature sensitivity seen for the rpb7–7 allele in rpb4Δ is not the result of reduction in the level of the mutant protein. The RNP Fold of Rpb7 Is an Important Functional Determinant–Rpb7 folds into two domains: an N-terminal truncated RNP fold and a C-terminal OB fold (Fig. 3A). Sequencing of the rpb7–7 allele revealed that it had four mutations (F70L, V72M, A123E, and S125T). The first two mutated residues are located on a conserved β-strand of the RNP fold, whereas the other two mutated residues are on the three-stranded antiparallel β-sheet insertion between strands B3 and B4 of the OB fold domain (Fig. 3A). The phenotype of rpb7–7 could be the consequence of a disrupted RNP fold or could be caused by alterations in the interactions mediated by the solvent-exposed C-terminal β-sheet or both. To distinguish between these possibilities, we swapped the N- and C-terminal domains of the mutant with the wild type Rpb7 to construct rpb7–7N allele carrying only the F70L and V72M mutations and the rpb7–7C allele carrying only the A123E and S125T mutations. Both of these mutant alleles could complement the l
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