The Human Mitochondrial Transcription Termination Factor (mTERF) Is FullyActive in Vitro in the Non-phosphorylatedForm

Abstract

The human mitochondrial transcription termination factor (mTERF) is a39-kDa protein that terminates transcription at the 3′-end of the 16 SrRNA gene and thereby controls expression of the ribosomal transcription unitof mitochondrial DNA. The transcription termination activity of human mTERFhas been notoriously difficult to study in vitro, and it has beensuggested that the activity of the protein is regulated by posttranslationalmodifications or by protein polymerization. We here characterize the activityof recombinant human mTERF expressed in insect cells. We observed that mTERFefficiently promotes sequence-specific termination in a completely recombinantand highly purified in vitro system for mitochondrial transcription.The termination activity has a distinct polarity, and we observed completetranscription termination when the mTERF-binding site is oriented in a forwardposition relative the heavy strand promoter but only partial transcriptiontermination when the binding site is in the reverse position. We analyzed thebiochemical characteristics of the active mTERF protein and found that it is astable monomer at physiological salt concentration. Structural analysis,including phosphostaining, two-dimensional electrophoresis, and electrospraymass spectrometry, detected no evidence of phosphorylation. We conclude thatthe monomeric human mTERF is fully active in its non-phosphorylated form andthat the protein does not require additional cellular factors to terminatemitochondrial transcription in vitro. The human mitochondrial transcription termination factor (mTERF) is a39-kDa protein that terminates transcription at the 3′-end of the 16 SrRNA gene and thereby controls expression of the ribosomal transcription unitof mitochondrial DNA. The transcription termination activity of human mTERFhas been notoriously difficult to study in vitro, and it has beensuggested that the activity of the protein is regulated by posttranslationalmodifications or by protein polymerization. We here characterize the activityof recombinant human mTERF expressed in insect cells. We observed that mTERFefficiently promotes sequence-specific termination in a completely recombinantand highly purified in vitro system for mitochondrial transcription.The termination activity has a distinct polarity, and we observed completetranscription termination when the mTERF-binding site is oriented in a forwardposition relative the heavy strand promoter but only partial transcriptiontermination when the binding site is in the reverse position. We analyzed thebiochemical characteristics of the active mTERF protein and found that it is astable monomer at physiological salt concentration. Structural analysis,including phosphostaining, two-dimensional electrophoresis, and electrospraymass spectrometry, detected no evidence of phosphorylation. We conclude thatthe monomeric human mTERF is fully active in its non-phosphorylated form andthat the protein does not require additional cellular factors to terminatemitochondrial transcription in vitro. Transcription of the heavy (H) strand of mitochondrial DNA (mtDNA) involvestwo overlapping transcription units(1Montoya J. Christianson T. Levens D. Rabinowitz M. Attardi G. Proc. Natl. Acad. Sci. U. S.A. 1982; 79: 7195-7199Crossref PubMed Scopus (206) Google Scholar,2Montoya J. Gaines G.L. Attardi G. Cell. 1983; 34: 151-159Abstract Full Text PDF PubMed Scopus (220) Google Scholar). One unit starts directlyupstream of the tRNAPhe gene and spans the tRNAPhe, 12 SrRNA, 16 S rRNA, and tRNAVal genes (initiation site H1). The othertranscription unit starts ∼100 bp further downstream (initiation site H2)at the boundary between the tRNAPhe and 12 S rRNA genes andproduces a single polycistronic RNA that encompasses almost the entire lengthof the heavy strand. The ribosomal transcription unit is transcribed at a muchhigher rate compared with the other transcription unit, and control of itsexpression is exerted at both the initiation and termination levels(3Gelfand R. Attardi G. Mol.Cell. Biol. 1981; 1: 497-511Crossref PubMed Scopus (154) Google Scholar,4Attardi G. Chomyn A. King M.P. Kruse B. Polosa P.L. Murdter N.N. Biochem. Soc.Trans. 1990; 18: 509-513Crossref PubMed Scopus (51) Google Scholar). A central role in thecontrol of termination has been attributed to the mitochondrial transcriptiontermination factor(mTERF), 1The abbreviations used are: mTERF, mitochondrial transcription factor;Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT,dithiothreitol; HSP, heavy strand promoter; IEF, isoelectric focusing; LSP,light strand promoter; MALDI, matrix-assisted laser desorption ionization;Ni2+-NTA, nickel-nitrilotriacetic acid; PVDF, polyvinylidenefluoride; TOF, time-of-flight. a 39-kDaprotein that binds to a 28-base pair region of mtDNA located within thetRNALeu(UUR) gene at a position immediately downstream of the 16 SrRNA gene (5Kruse B. Narasimhan N. Attardi G. Cell. 1989; 58: 391-397Abstract Full Text PDF PubMed Scopus (225) Google Scholar,6Daga A. Micol V. Hess D. Aebersold R. Attardi G. J. Biol. Chem. 1993; 268: 8123-8130Abstract Full Text PDF PubMed Google Scholar). Previous characterizationof recombinant mTERF expressed in bacteria or in coupled in vitrotranscription and translation systems produced proteins with sequence-specificDNA binding activity but no transcription termination activity(7Fernandez-Silva P. Martinez-Azorin F. Micol V. Attardi G. EMBO J. 1997; 16: 1066-1079Crossref PubMed Scopus (140) Google Scholar). These observationsindicated that mTERF requires other cellular factors and/or aposttranslational modification to efficiently terminate transcription.Furthermore, a recent study of recombinant rat mTERF suggested thatphosphorylation of four amino acids is required for transcription terminationactivity (8Prieto-Martin A. Montoya J. Martinez-Azorin F. Nucleic Acids Res. 2004; 32: 2059-2068Crossref PubMed Scopus (26) Google Scholar). Alternatively,mTERF activity may be regulated by its polymerization state, because nativehuman mTERF is present in HeLa mitochondrial lysates in two forms, an activemonomer and an inactive polymer(9Asin-Cayuela J. Helm M. Attardi G. J. Biol. Chem. 2004; 279: 15670-15677Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Native mTERF has a hightendency to polymerize, thereby explaining a dramatic loss of activity duringpurification. We here characterize mTERF-dependent transcription termination in a highlypurified and recombinant in vitro system for mitochondrialtranscription (10Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Nat.Genet. 2002; 31: 289-294Crossref PubMed Scopus (454) Google Scholar). Previousinvestigations of mTERF function were performed in partially purifiedmitochondrial extracts, and the possible contribution of yet to be identifiedcellular factors was therefore difficult to address. Because bacterialexpression in the past had failed to generate an active human protein, weexpressed recombinant mTERF in insect cells using the baculovirus system. Theadvantages of this eukaryotic expression system are the abundant expression ofrecombinant protein combined with the possibility of co-translational andposttranslational modifications, including N-glycosylation andphosphorylation(11Davidson D.J. Castellino F.J. Biochemistry. 1991; 30: 6165-6174PubMed Google Scholar, 12Davidson D.J. Bretthauer R.K. Castellino F.J. Biochemistry. 1991; 30: 9811-9815Crossref PubMed Scopus (46) Google Scholar, 13Ku N.O. Omary M.B. Exp.Cell Res. 1994; 211: 24-35Crossref PubMed Scopus (46) Google Scholar, 14Duhe R.J. Farrar W.L. J.Biol. Chem. 1995; 270: 23084-23089Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).We found that baculovirus-expressed human mTERF efficiently terminatestranscription in vitro and displays distinct strand specificity. ThemTERF protein does not require other cellular factors for its terminationactivity, and a comprehensive structural analysis revealed that mTERF is astable monomer in solution that actively promotes transcription termination inits non-phosphorylated form. Expression and Purification of Recombinant Human mTERF—Thegene encoding mTERF (GenBank™ accession number BC000965) was amplifiedfrom cDNA by PCR and cloned it into the pBacPAK9 (Clontech). During thecloning the leader peptide was deleted, a Met residue was added to the Nterminus, and a His6 tag was added to the C terminus.Autographa californica nuclear polyhedrosis virus, recombinant formTERF, was prepared as described in the BacPak manual (Clontech). For proteinexpression, Spodoptera frugiperda (Sf9) cells grown in suspensionwere infected with the virus (5–10 plaque-forming unit/cell) andincubated for 60–72 h at 27 °C in SF 900 II medium withl-glutamine (Invitrogen) complemented with 2% fetal calf serum and1% PEST antibiotic mixture (Invitrogen). Cells were then pelleted down, washedin phosphate-buffered saline, snap-frozen in liquid nitrogen, and stored at–80 °C. The recombinant mTERF protein was purified by Ni2+-NTAchromatography. The frozen cell pellets were resuspended in 10 ml of lysisbuffer (25 mm Tris-HCl (pH 8.0), 10 mm2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 2μm pepstatin, 0.6 μm leupeptin, and 2mm benzamidine) and incubated on ice for 20 min. The cells werehomogenized with 15 strokes of a tight-fitting pestle in a Dounce homogenizer,and the homogenate was brought to 15 ml with lysis buffer. After the additionof 3 ml of 5 m NaCl, the solution was incubated at 4 °C for 45min in an orbital shaker. The insoluble material was removed by centrifugationat 100,000 × g for 40 min at 4 °C. The supernatantcontaining mTERF was passed 10 times through an 18-gauge needle to shear DNA.One volume of buffer A (25 mm Tris-HCl (pH 8.0), 1 mNaCl, 10 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonylfluoride, 2 μm pepstatin, 0.6 μm leupeptin, and 2mm benzamidine) supplemented with 20 mm imidazole wasadded to the supernatant. The lysate was mixed with Ni2+-NTASuperflow resin (Qiagen) previously equilibrated in buffer A supplemented with10 mm imidazole (1 ml Ni2+-NTA resin per 400 ml of cellculture). The mixture of supernatant and resin was incubated at 4 °C for 1h in an orbital shaker, and the beads were then collected by centrifugationfor 10 min at 1,500 × g. After removal of the supernatant(flow-through), 10 resin volumes of buffer A supplemented with 40mm imidazole were added to the resin. After 15 min of incubation,the mixture was centrifuged for 10 min at 1,500 × g, afterwhich the supernatant (wash) was removed and the resin transferred into aPoly-Prep® chromatography column (Bio-Rad). The resin wasincubated for 15 min with 12 ml of elution buffer (buffer A supplemented with250 mm imidazole), and the mTERF protein was subsequently eluted in1-ml fractions that were snap frozen in liquid nitrogen and stored at–80 °C. Gel Filtration Chromatography—The gel filtrationchromatography of Ni2+-NTA-purified mTERF was performed asdescribed (9Asin-Cayuela J. Helm M. Attardi G. J. Biol. Chem. 2004; 279: 15670-15677Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) with thefollowing modifications. The column (Superose® 12; AmershamBiosciences) was equilibrated in running buffer (25 mm HEPES (pH7.6), 5 mm MgCl2, 0.5 mm EDTA, 1mm dithiothreitol, 10% glycerol, 0.1% Tween 20, and 500mm KCl) at a flow rate of 0.3 ml/min. Following the instructions ofthe column's manufacturer, a calibration curve was prepared with blue dextran2000, ferritin (440 kDa), catalase (232 kDa), ovalbumin (47 kDa), and insulin(5.8 kDa), all from Amersham Biosciences (except for insulin from Sigma,catalog number I9278). The protein samples were dialyzed against gelfiltration running buffer prior to injection. The injected sample volume was 1ml, the flow rate was 0.4 ml/min at 4 °C, and 0.2-ml fractions werecollected. Fractions were analyzed by SDS-PAGE and Coomassie Blue staining.The mTERF-containing fractions were snap-frozen in liquid nitrogen and storedat –80 °C. The mTERF protein concentration was determined withBradford reagent (Sigma). In Vitro Transcription Assay—The termination activity ofmTERF was tested in a fully reconstituted in vitro transcriptionsystem described previously(10Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Nat.Genet. 2002; 31: 289-294Crossref PubMed Scopus (454) Google Scholar). Four different DNAtemplates were used. To construct pHSP1, a DNA fragment corresponding to bp499–742 of human mtDNA (containing both HSPs) was cloned into pUC18. Toconstruct pHSP1-TERM, a fragment corresponding to bp 3063–3594 of humanmtDNA (containing the binding site for mTERF, between nucleotides 3229 and3256) was cloned immediately downstream of HSP1 fragment in pHSP1. For thegeneration of pHSP1-MRET, the human mtDNA 3063–3595 fragment was clonedinto the same position as in pHSP1-TERM, but in the reverse orientation. ForpLSP-MRET, a LSP-containing fragment spanning bp 477–1 of human mtDNAwas cloned into pUC18 followed by the mTERF-binding site fragment(3594–3063) immediately downstream. After linearization (pHSP1-TERM andpHSP1-MRET with BmrI and pLSP-MRET with EcoRI), the plasmid constructs wereused to measure promoter-specific transcription in a runoff assay as describedpreviously (10Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Nat.Genet. 2002; 31: 289-294Crossref PubMed Scopus (454) Google Scholar). The proteinconcentrations used in the in vitro transcription experiments were250 fmol of His-POLRMT, 500 fmol of TFB2M, 2.5 pmol of TFAM and, whenindicated, variable amounts of mTERF or an equivalent volume of buffer. For the pulse-chase experiment, pHSP-TERM was used as template, and thein vitro transcription reaction was scaled up to a final volume of100 μl. The in vitro transcription reaction contained 0.3μm [α-32P]UTP (100 μCi), and after 5 min ofincubation at 32 °C cold UTP was added to a final concentration of 2.5mm. Samples (25 μl) were collected 0, 30, 60, and 90 min afterthe addition of UTP, and the transcription products were analyzed as describedpreviously (10Falkenberg M. Gaspari M. Rantanen A. Trifunovic A. Larsson N.G. Gustafsson C.M. Nat.Genet. 2002; 31: 289-294Crossref PubMed Scopus (454) Google Scholar). Phosphoprotein Blot Staining—The indicated amounts ofrecombinant mTERF, ovalbumin, and bovine serum albumin were separated byelectrophoresis on a 12.5% SDS-polyacrylamide gel, and the proteins wereblotted to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane wassubsequently stained with the Pro-Q® Diamond phosphoproteinblot stain kit (Molecular Probes) following the instructions of themanufacturer. The stained bands were revealed by direct UV lighttransillumination and photographed with a charge-coupled device camera, modelICD-42E (Ikegami Tsushinki Co.) Two-dimensional Electrophoresis—The mTERF protein wasseparated by isoelectric focusing (IEF) in the first dimension and by SDS-PAGEin the second dimension. For the analysis an mTERF protein purified by gelfiltration over a Superose® 12 column and dialyzed against arunning buffer (25 mm HEPES (pH 7.6), 5 mmMgCl2, 0.5 mm EDTA, 10% glycerol, 0.1% Tween 20, 1mm DTT, and 100 mm KCl) was used. IEF was carried outessentially according to the instructions of the manufacturer (AmershamBiosciences). The IEF strips (Immobiline™ DryStrip pH 7–11 NL, 7cm) were rehydrated overnight in 200 μl of DeStreak®rehydration solution containing 1% of IPG pH 7–11 NL buffer. Twentymicroliters of mTERF (1pmol/μl) were mixed with 69.5 μl of buffer 2 m and 10 μl of and 0.5 μl of IPG pH 7–11 NL and the the IEF in a sample The IEF on an IEF unit at 20 °C with per using the following 1 and 200 2 and and and 4 and and For the 20 μl of (1 were incubated with μl of buffer and 4 μl of calf (1 at °C for a the calf with 4 μl of After the samples in DTT, and IPG buffer and the as described After IEF, the strips were incubated for 15 min at in 10 ml of solution mm m glycerol, 2% and supplemented with 10 DTT, followed by a second for in solution supplemented with was on of a 12.5% SDS-polyacrylamide that with a solution of and blue in buffer. After electrophoresis, the were Coomassie Blue or as protein was used as a with desorption analysis of proteins was carried with an II from The samples were prepared asdescribed previously V. D. T. T. Gustafsson C.M. Proc. Natl. Acad. Sci. A. PubMed Scopus Google were in the at an and a of for were with the from by using the For spectrometry, 20 μl mTERF was on a equilibrated in 0.1% The column was for 2 min with 0.1% at a of 200 and mTERF was subsequently eluted with a and 0.1% at a flow rate of The protein samples were analyzed with an using for the calibration with 100 pmol of of the was the with the The the were as Recombinant mTERF Transcription in a in Vitro Transcription that mTERF activity is regulated by or We to the for mTERF vitro and therefore a recombinant baculovirus form of the human mTERF protein with Recombinant mTERF was expressed cells and purified by Ni2+-NTA chromatography and gelfiltration to We mTERF-dependent transcriptiontermination in an in vitro system for mitochondrial with highly purified and recombinant transcription factor RNA and transcription factor For we used DNA containing followed by site in the or reverse orientation. We experiments in but on two different to of the transcription termination activity on the reverse In the of mTERF, the mitochondrialtranscription the 2 in We added amounts of mTERF and observed of an additional with the for an at the mTERF-binding The recombinant mTERF highly and we observed termination at a of mTERF to DNA 4 The mTERF termination activity was of of the mTERF-binding the mTERF-binding in the same as the we failed to termination at the mTERF the mTERF-binding site in the reverse observed almost termination of transcription at a of of mTERF to DNA The mTERF protein has been previously to B. Narasimhan N. Attardi G. Cell. 1989; 58: 391-397Abstract Full Text PDF PubMed Scopus (225) Google J. Helm M. Attardi G. J. Biol. Chem. 2004; 279: 15670-15677Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The is when mTERF binds to its binding site and both the the mTERF-binding site are in the same orientation. In vitro transcription we that the was on the pHSP-TERM relative We the addition of amounts of mTERF to two To further characterize the mTERF protein, we analyzed transcriptiontermination with the followed by the mTERF-binding site in the as in We observed and termination but no of the The observed were had been observed with the we to the observed was termination of transcription and not only between two we performed a with the pHSP-TERM After a of added an of cold UTP and followed the for 90 min. mTERF RNA we thatthe of to the of by the We the for up to 90 min of in the between and runoff that the in the of mTERF to transcription termination and not to RNA Recombinant mTERF a mTERF protein cells has been previously to in two as an active monomer and as an inactive We determined of recombinant mTERF with gel Superose® The mTERF protein eluted as single an of corresponding to of the protein Because the gel analysis had at salt m KCl) that a we fractions from the gel the material against a buffer with m the gel the eluted as a monomer We that recombinant mTERF active monomeric and does not to the same previously for The Recombinant mTERF of rat mTERF and its by to the of amino in We first used a Diamond binds to the and has been to as 2 of on PVDF T. B. 2004; PubMed Scopus (26) Google Scholar). We separated mTERF in on a SDS-polyacrylamide gel ovalbumin (containing two and used as a and bovine serum albumin as a After a PVDF we stained the proteins with Diamond We of mTERF, when 3 of it was but with 1 of Phosphoprotein is a and and Diamond on the same samples revealed T. B. 2004; PubMed Scopus (26) Google Scholar). We with and therefore of mTERF two-dimensional gel For dimension IEF we used of in the running buffer to a by of In dimension we separated the mTERF protein with SDS-PAGE and stained Blue we detected a that in with the (pH and of II and be observed and the was were by The mTERF protein is to as a to the of immediately or the We not to the the for form of In a second of we after a we the mTERF protein sample to but the In two experiments we observed at same position. The of mTERF in the was not the of mTERF, against the In a final to amino mTERF by spectrometry, a that of the of a peptide with the protein of the protein was determined by The determined for the 0.6 with that the mTERF with an additional of and with the analysis revealed protein at with a of The in of relative to the at is with a between and of an We used here a highly purified and recombinant transcription system recombinant human mTERF in vitro. system the of and that mTERF does not require additional factors present to actively transcription previously with the protein, recombinant mTERF terminatestranscription of both and J. J.Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The purified is highly and we transcription termination protein the concentration of the DNA transcription termination at of mTERF relative to that of the DNA from study is that non-phosphorylated mTERF is active We phosphostaining, and analysis to the active mTERF protein is not of that a of mTERF is and is for the termination activity observed because the mTERF protein promotes almost completetranscription termination at a of protein template, this possibility human mTERF does not require to in vitro, we not to a role in the of mTERF activity of mTERF and the protein or the DNA specificity. Previous of the mTERF protein the protein does not only terminate transcription at the but transcription initiation at B. Narasimhan N. Attardi G. Cell. 1989; 58: 391-397Abstract Full Text PDF PubMed Scopus (225) Google J. Helm M. Attardi G. J. Biol. Chem. 2004; 279: 15670-15677Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The was when mTERF binds to its site and both the the mTERF-binding site are in the same orientation. We failed to of transcription in reconstituted a between the recombinant protein in this transcription termination factors been from different including Polosa P. M. P. Res. PubMed Scopus Google M. Polosa P.L. F. P. Nucleic 31: PubMed Scopus Google and analyzed here the characteristics of factor in a fully in vitro transcription we system and with to further the transcription The and for to the expression of recombinant TFAM and of on two-dimensional was We for to