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Purification and Characterization of an RNA Dodecamer Sequence Binding Protein from Mitochondria of Saccharomyces cerevisiae
Biochemical and Biophysical Research Communications 261, 740 –745 (1999) Article ID bbrc.1999.1085, available online at http://www.idealibrary.com on
Purification and Characterization of an RNA Dodecamer Sequence Binding Protein from Mitochondria of Saccharomyces cerevisiae Huilin Li and H. Peter Zassenhaus 1 Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
Received July 8, 1999
Saccharomyces cerevisiae mitochondrial mRNAs terminate at their 3* ends with a conserved dodecamer sequence, 5*-AAUAA(U/C)AUUCUU-3*. We have identified a nuclear-encoded protein (DBP) which specifically binds to the dodecamer sequence and have purified it to apparent homogeneity by RNA affinity chromatography. DBP consists of a single polypeptide of 55 kDa and binds to its RNA substrate with a 1:1 stoichiometry. Scatchard analysis determines that K d is 0.93 nM for the canonical dodecamer sequence (5*AAUAAUAUUCUU-3*) and 0.46 nM for the only naturally occurring variant (5*-AAUAACAUUCUU-3*) unique to oli1 gene. Based on the studies using mutant oligonucleotides, DBP appears to recognize primarily the nucleotide sequence of an RNA rather than its potential secondary structure. © 1999 Academic Press
Previous work in our laboratory identified a protein from mitochondria of Saccharomyces cerevisiae that specifically binds to a highly conserved dodecamer sequence, 59-AAUAA(U/C)AUUCUU-39, which is found at the 39 ends of all mitochondrial mRNAs (1, 2). This sequence is encoded within multi-cistronic pre-mRNAs and appears to be a site for specific cleavage event that occurs a few nucleotides downstream of the dodecamer sequence (2). Since mitochondrial mRNAs in yeast are not polyadenylated, mature mRNAs terminate with the dodecamer sequence. We identified a protein (termed DBP for dodecamer binding protein) that bound to that sequence whether located internally within an RNA or at its 39 terminus (3). Although direct experimental evidence for the functions of DBP is not yet available, it is likely that it 1
To whom correspondence should be addressed. Fax: (314) 7733403. E-mail: [email protected]. Abbreviations used: DBP, dodecamer sequence binding protein; EMSA, electrophoretic mobility shift analysis; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
plays an important role in pre-mRNA processing and stabilization of mRNAs based upon the analysis of strains with mutant dodecamer sequences. Point mutations within the v intron dodecamer sequence eliminate endoribonucleolytic processing of v-containing RNAs at that site (4 – 6). RNA oligonucleotides containing the mutant v dodecamer sequence also show greatly reduced binding to DBP in vitro (3), suggesting that DBP plays a role in directing site-specific cleavage of that RNA. Deletion of the dodecamer site plus some flanking sequence associated with the var1 gene results in aberrantly processed var1 transcripts that appear to be more rapidly degraded than their wild-type counterpart (4). This observation together with the fact that most mitochondrial mRNAs terminate with a dodecamer sequence (2) suggests that DBP binding to mature mRNAs is important for their stability in vivo. In vitro, we have shown that DBP binding to the 39 region of an RNA protects it from digestion by an NTP-dependent 39 exoribonuclease that is found in yeast mitochondria (3). This enzyme appears to function in the turnover of mitochondrial RNAs (7, 8), and binding of DBP to the 39 ends of mitochondria mRNAs may therefore regulate mRNA stability by regulating the access of that enzyme to mRNAs. To further delineate the role of DBP in RNA processing and mRNA function, we have purified DBP and characterized its interaction with RNA substrates of differing sequence. Unlike most other sequencespecific RNA binding proteins (9, 10), DBP binding to its substrate does not appear to require secondary structure in the RNA that is based upon base-pairing. Rather, DBP appears to recognize the primary sequence directly in a single-stranded RNA. EXPERIMENTAL PROCEDURES Purification of DBP. A1237 m161 (Mata ad1 ura3 leu2 nuc1-1 : : LEU2) was grown to late log phase in YPGal (1% yeast extract, 2% Bacto peptone, 4% galactose) at 30°C with vigorous
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shaking. About 1.5 kg wet weight of cells was harvested from 60 liters of culture and mitochondria were prepared as described previously, except that they were not gradient purified (11). The mitochondrial preparation was suspended in 150 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 0.5 M KCl, 5 mM MgCl 2, 1 mM EDTA, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 0.1% Triton X-100) and sonicated on ice for 10 min. The suspension was clarified by centrifugation at 10,000 3 g for 20 min. The pellet was processed two more times as above (using 100 ml lysis buffer each time) and the supernatants were combined and centrifuged at 100,000 3 g for 1 h. The resulting supernatant (about 360 ml) was dialyzed overnight against Buffer A (10 mM Tris–HCl, pH 7.5, 5 mM MgCl 2, 0.1 mM EDTA, 2 mM DTT, 10% glycerol) plus 0.1 M KCl and applied to a heparin-Sepharose column equilibrated in Buffer A containing 0.15 M KCl. After washing with 5 column volumes of the same buffer, DBP was eluted with a 500 ml gradient of 0.15– 0.5 M KCl in Buffer A (Fig. 1A and Table 1). Fractions containing DBP binding activity were pooled and concentrated three fold by dialysis against 20% polyethylene glycol (MW 20,000) in Buffer A and the concentration of KCl adjusted to 0.2 M KCl. DBP binding activity was further purified by RNA affinity chromatography. A chimeric RNA/DNA oligonucleotide was synthesized so that the first 30 residues (from the 59 end) were poly(dA) which were then followed in the oligonucleotide by the dodecamer containing RNA sequence 59-AUAAAAUAACAUUCUUAAUU-39. The chimeric oligonucleotide (550 mg) was annealed to a poly(dT)-cellulose support (Type 7, Pharmacia; 500 mg) and equilibrated with Buffer A plus 0.2 M KCl. The binding capacity of this column was approximately 0.1 nMole of DBP. A portion of the dialysate (;20%) was applied to the RNA affinity column and, after washing with 10 column volumes of Buffer A containing 0.2 M KCl, DBP was eluted with a 50 ml linear gradient of KCl (0.2– 0.6 M in Buffer A). The amount of protein in each step of the purification was determined using the Pierce BCA Protein Assay Reagent and bovine serum albumin (BSA) as a standard. The amount of protein after the final step was estimated from Coomassie Brilliant Blue staining of SDS– PAGE fractionated samples in comparison to the staining intensity of known amounts of BSA. Preparation of different RNA substrates. RNA oligonucleotides were gel-purified for selection of full-length species and the amount of purified oligonucleotide quantified by measuring UV absorbence at 260 nm. The nomenclature and sequence of the oligonucleotides used here were: (1) Var, 59-AUAAAAUAAUAUUCUUAAUU-39, which contained the canonical dodecamer sequence as underlined plus the flanking 4 nucleotides on each side as found at the dodecamer site associated with the var1 gene; (2) a set of sequence variants, Var 3U3 C, Var 3,4UA3 AU, Var 6U3 C, Var 10C3 A, where the subscript denotes the nucleotide position within the dodecamer sequence that was altered followed by the sequence change; and (3) NS, 59-AUAUAUUAAUAUAUAUACUU-39, an oligonucleotide with the same sequence composition as Var which was used as a control for non-specific binding. Oligonucleotides were 59-end labeled with T4 polynucleotide kinase and [g- 32P]ATP (Amersham) according to instructions supplied by the enzyme’s manufacturer (New England Biolabs). Quantification of DBP binding activity by electrophoretic mobility shift assay (EMSA). DBP binding activity was measured in a 15 ml reaction mixture containing 10 mM Tris–HCl, pH 7.5, 200 mM KCl, 1 mM EDTA, 2 mM DTT, 0.1 mg/ml tRNA (Sigma), 10% glycerol, and 32 P-labeled oligonucleotide Var. The reaction mixture was incubated at 4°C for 15 min and then fractionated by PAGE as described (3). Radioactivity was visualized by scanning the gel with a Molecular Dynamics PhosphorImager. Quantitative measurement of DBP binding activity was performed using saturating amounts (10 nM) of oligonucleotide Var 6U3 C. The amount of DBP was calculated based on the 1:1 stoichiometry between DBP and RNA.
UV cross-linking. UV cross-linking was performed as previously described (3). For in situ UV cross-linking, the complex between a 32 P-labeled Var oligonucleotide and purified DBP was first fractionated on a native gel and then the gel piece containing the labeled complex was irradiated with 256 nm UV light for 10 min (12 joules). The irradiated complex was eluted from the crushed gel by soaking in Laemmli loading buffer (12) and fractionated by SDS–PAGE. Determination of dissociation constants (K d ) for the binding between DBP and RNA oligonucleotides. A series of binding reactions containing a fixed concentration of DBP (50 pM) were performed in which the concentration of the labeled RNA oligonucleotide varied from 0.05 nM to 50 nM. The amount of bound and free oligonucleotide was determined by EMSA. The equilibrium dissociation constant was calculated by Scatchard analysis. Glycerol gradient sedimentation. 100 ml of a reaction mixture containing 32P-labeled oligonucleotide Var and purified DBP was layered on top of a 4.0 ml 10 –35% (v/v) glycerol gradient in Buffer A containing 0.2 M KCl. The gradient was centrifuged at 40,000 rpm at 4°C for 21 h in a Beckman SW 50.1 rotor. After centrifugation, 100 ml fractions were collected from the bottom of the gradient and assayed for the DBP–RNA complex by mobility shift assay. Protein standards of known molecular weight were centrifuged in a parallel gradient. Gel filtration. Gel filtration of complexes between DBP and 32Plabeled RNA was performed on Sephacryl S-200 (0.8 3 30 cm) equilibrated in Buffer A containing 0.2 M KCl. Elution of DBP–RNA complexes was determined by EMSA. Blue Dextran was used for determination of the void volume (V o). Ferguson analysis. Reaction mixtures containing 32P-labeled RNA-DBP complexes were electrophoresed on 4%, 6%, 8% and 10% native polyacrylamide gels and the relative electrophoretic mobilities (Rm) measured relative to the dye front (bromophenol blue). Molecular weights were calculated from the Ferguson Plot as described (16).
RESULTS Purification of DBP. DBP was purified from a mitochondrial extract by heparin-agarose chromatography (Fig. 1, Panel A) followed by RNA affinity chromatography (Fig. 1, Panel B). The two sequential chromatographic steps purified DBP by about 10,000fold with a 30% yield (Table 1). Based upon the amount of DBP found in whole cell lysates, it was estimated that each yeast cell contains about 1000 to 2000 DBP molecules. When the peak fraction from the eluate of the RNA affinity column was analyzed by SDS–PAGE and silver staining, the major polypeptide observed had an apparent molecular weight of 55 kDa (Fig. 1, Panel C). Of the proteins eluting from the RNA affinity column, only p55 co-fractionated with DBP activity (data not shown). Previously, we had shown that UV irradiation of reaction mixtures containing a mitochondrial extract and 32P-labeled Var gave rise to three 32P-labeled polypeptide with apparent molecular weights of 70, 55, and 19 kDa (3). When the purified DBP preparation was similarly analyzed, we likewise observed the formation of those three cross-linked species (Fig. 1, Panel D, lane 1), although p55 was always the major crosslinked species. The formation of those species was inhibited by the inclusion of 100 fold excess unlabeled Var (Panel D, lane 2) but not by the non-specific RNA
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FIG. 1. Purification of DBP from yeast mitochondria. DBP was purified by sequential chromatography of a mitochondrial extract through heparin-Sepharose (Panel A) and an RNA affinity column (Panel B). Solid circles, relative amounts of DBP as determined by mobility shift assay and quantification of the bound complex by densitometry of the gel using the Molecular Dynamics PhosphorImager; open circles, concentration of KCl. Panel C, SDS–PAGE of a peak fraction from the eluate of the RNA affinity column. Lane 1, sample buffer blank; lane 2, a peak fraction from the RNA affinity chromatography. Only the p55 polypeptide co-fractionated with binding activity eluted from the RNA affinity column. The mobilities of molecular size markers (in kilodaltons) are indicated on the left of the gel. Panel D, UV crosslinking of p55 to RNA oligonucleotides. In lanes 1–3, purified DBP was incubated with RNA oligonucleotides, illuminated by UV light and then analyzed by SDS–PAGE. Lane 1, 32P-labeled Var only; lane 2, 32P-labeled Var plus 100-fold molar excess of unlabeled Var; lane 3, 32P-labeled Var plus 100-fold molar excess of NS. In lane 4, the complex between 32P-labeled Var and purified DBP was first electrophoretically fractionated on a native gel before exposure to UV light in situ. The UV-irradiated complex was eluted from the gel and then analyzed by SDS–PAGE.
oligonucleotide, NS (Panel D, lane 3). When DBP/RNA complexes were first fractionated by native gel electrophoresis before UV irradiation in situ, followed by SDS–PAGE of the eluted material, p55 was again the major cross-linked species observed (Panel D, lane 4) with only minor amounts of p70 and p19 detectable after long exposures of the gel. We interpret these results to indicate that p55 alone accounts for the dodecamer-site binding activity present in mitochondrial lysates.
towards the six different oligonucleotides mirrored the behaviour of the activity present in the mitochondrial extract (Fig. 2). As noted before (3), DBP binding to Var 6 U3 C was greater than towards the canonical dodecamer sequence (Var). In contrast, both the purified and crude activities showed much less binding to the sequence variants and none at all to the NS oligonucleotide. Although Fig. 2 shows a stronger binding of Var 3U3 C to purified DBP than to the activity in a crude mitochondrial lysate, a detailed analysis using
Binding specificity of the purified DBP. To determine whether the purified DBP had the same binding specificity as the activity previously described in mitochondrial extracts (3), a set of six 32P-labeled RNA oligonucleotides of differing sequences were synthesized for comparison of binding activities. Overall, we found that the binding activity of the purified DBP
TABLE 1
Purification of the Dodecamer-Site Binding Protein Step
Protein (mg)
DBP (nmole)
Yield (%)
Mitochondria Mitoplasts 100,000 3 g Supernatant Heparin-Sepharose RNA Affinity
9,000 5,000 3,500 100 ;0.1
— — 2.0 1.1 0.6
— — 100 53 30
FIG. 2. Binding specificity of the purified DBP compared to the activity present in a mitochondrial extract. A mitochondrial extract (lanes 1– 6) and the purified DBP (lanes 7–12) were incubated with a set of six 32P-labeled RNA oligonucleotides of differing sequence. The sequences of RNA oligonucleotides (indicated above each lane) used in the binding reactions are described in the “Experimental Procedures.” The binding reaction was carried out under standard conditions containing 1 nM oligonucleotides. The labeled complexes were analyzed by mobility shift assay and visualized by a Molecular Dynamics PhosphorImager.
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one associated with the oli1 gene (1, 2). Here, a U to C replacement occurs at position 6 of the canonical dodecamer sequence. To determine whether that sequence change affected DBP binding, we compared the binding affinity of DBP towards oligonucleotide Var vs Var 6U3 C. By a Scatchard analysis, the K d using Var 6U3 C was found to be 0.46 nM whereas with Var it was 0.93 nM (Fig. 3).
FIG. 3. Scatchard analysis for the measurement of DBP binding affinity to RNA oligonucleotides. A series of binding reactions containing a fixed concentration of DBP (50 pM) were performed in which the concentration of the labeled RNA oligonucleotides, Var and Var 6U3 C, varied from 0.05 nM to 50 nM. The bound and free oligonucleotides were fractionated by EMSA and quantified on a Molecular Dynamic PhosphorImager. The dissociation constants (K d) of DBP for Var or Var 6U3 C were determined by Scatchard analysis.
competition assays revealed that, as with crude lysate, purified DBP bound weakly to Var 3U3 C (data not shown). These results indicate that purified DBP accounts for the binding activity observed in mitochondrial lysates. Binding affinity. All dodecamer sites in the mitochondrial genome have the same sequence except the
Stoichiometry of DBP–RNA complex. To determine whether DBP bound RNA as a monomer or higherorder polymer, we measured the apparent size of the complex formed between purified DBP and a 20-base long RNA oligonucleotide. Three different techniques were employed so as to reduce the likelihood that the measured size was confounded by conformational artifacts. Whether by glycerol gradient sedimentation (Fig. 4, Panel 1), gel filtration (Fig. 4, Panel 2), or gel electrophoresis combined with a Ferguson analysis (Fig. 4, Panel 3), the observed size of the complex was consistently found to be between 50 – 60 kDa. Since the purified DBP itself has a molecular weight of about 55 kDa, these results suggest that DBP bind RNA as a monomer. We also determined that complexes contained only a single RNA molecule (data not shown), indicating that the stoichiometry of DBP binding to RNA was 1:1. The requirement of secondary structure in the RNA for binding activity. In many cases, the secondary structure of an RNA substrate plays an important role for site-specific binding (16, 17). The dodecamer se-
FIG. 4. Apparent size of the DBP–RNA complex. The apparent size of the complex between purified DBP and 32P-labeled Var was estimated by glycerol gradient sedimentation (Panel A), gel filtration (Panel B), and native gel electrophoresis in a Ferguson Plot analysis (Panel C). The standard marker proteins used in these experiments were b-AMS (b-amylase, 200 kDa), YAD (yeast alcohol dehydrogenase, 150 kDa), BSA (bovine serum albumin, 66 kDa), CA (carbonic anhydrase, 29 kDa) and cyt-C (cytochrome C, 12.5 kDa). DBP–RNA complexes were detected by electrophoresis on a native polyacrylamide gel. (Panel C) 32P-labeled DBP–RNA complexes were fractionated on 4%, 6%, 8% and 10% native polyacrylamide gels in parallel to the protein standards. Relative electrophoretic mobilities (Rm) were measured with respect to the dye front (bromophenol blue) and the Ferguson Plot was constructed according to Ferguson. The solid bars indicate the position of the DBP–RNA complex. 743
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FIG. 5. The role of potential secondary structure of the dodecamer sequence for DBP binding. The potential secondary structure of the canonical dodecamer sequence in the RNA oligonucleotide Var is diagrammed at the left. The dodecamer sequence resides within the shaded portion with each residue of the binding site numbered starting from the 59 end. The sequence alterations are illustrated at the middle of the diagram. Var 1,2 contained substitution of U’s for A’s at positions 1 and 2; In Var 11,12, A’s replaced U’s at positions 11 and 12; and Var 1,2/11,12 was synthesized so as to contain both sets of above sequence changes. 32P-labeled Var and the different modified versions were reacted separately with purified DBP in the standard binding reactions and analyzed by EMSA, which was then visualized by a Molecular Dynamics PhosphorImager.
quence is nearly palindromic (AAUAAU\AUUCUU). This feature may allow an oligonucleotide to form a stem-loop structure as illustrated in the left diagram of Fig. 5 for oligonucleotide Var. The dodecamer sequence is highlighted within the shaded box. Residues within the binding site are numbered starting from the 59 end. To test whether secondary structure is required for DBP binding, oligonucleotides were synthesized that contained sequence changes which were predicted to disrupt potential base pairing. An oligonucleotide was synthesized wherein the two adenosine residues at positions 1 and 2 were replaced by uridines; likewise, another oligonucleotide was made replacing the two uridine residues at positions 11 and 12 with adenosine residues (Fig. 5). A third oligonucleotide was also made containing both sets of the above sequence changes so that potential base pairing would be restored because of compensatory base substitutions. If secondary structure was required for DBP binding, we predicted that this last oligonucleotide would show binding activity whereas the first two would not. As shown in Fig. 5, Panel B, DBP failed to bind to any of these modified oligonucleotides, suggesting that secondary structure within the dodecamer site is not necessary for binding activity. DISCUSSION Several lines of evidence indicated that p55 was responsible for the DBP activity detected in a mitochondrial lysate. First, purified p55 shared the same binding specificity as the DBP activity in a crude lysate. Second, p55 was the only polypeptide to co-fractionate with DBP activity by RNA-affinity chromatography. Third, both p55 and crude DBP gave a similar spec-
trum of labeled adducts when UV cross-linked to a 32 P-labeled RNA oligonucleotide. In each case, the major adduct had an apparent molecular weight of about 55 kDa, similar to that of purified p55. The origin of the minor adducts of approximately 19 and 70 kDa is unclear at the moment. Finally, the native size of the binding complex was between 50 – 60 kDa, again similar to that of purified p55. Understanding the basis for the specific interaction between DBP and its RNA substrate requires knowing the stoichiometry of the complex. Many site-specific DNA binding proteins bind to their substrate as dimers when the DNA sequence within the binding site itself is symmetric. To determine the number of DBP molecules in a complex with its RNA substrate, the size of the complex was measured by three independent experimental approaches. All three (glycerol gradient sedimentation, gel filtration, and native gel electrophoresis) gave the same result: the size of the native complex was between 50 and 60 kDa, similar to the apparent molecular weight (55 kDa) of DBP. From these results we conclude that DBP binds to its RNA substrate as a monomer. Furthermore, from a determination of the number of RNA molecules in a single complex with DBP, it appears that one DBP molecule binds to one RNA substrate molecule. When present in mitochondrial RNAs, the dodecamer site is not tandemly duplicated. Given a 1:1 stoichiometry between DBP and RNA in a complex, it appears that DBP binding in vivo to either pre-mRNAs or mature mRNAs would not exhibit cooperativity. Consequently, the regulation of DBP binding to premRNAs and mRNAs may rely on either mass action, which would demand large changes in the concentration of DBP to change the relative amount of RNA in
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complex with DBP, or possibly on post-translational modification, such as phosphorylation. An increasing number of RNA binding proteins have been shown to regulate their binding affinity via phosphorylation (14). We have demonstrated that treatment of DBP with alkaline phosphatase abolishes specific, high affinity RNA binding (manuscript in preparation), suggesting that reversible phosphorylation may play a role in the regulation of DBP binding activity. Each yeast cell contains between 1000 to 2000 DBP molecules, which is close to the number of mitochondrial mRNA molecules present within the organelle (15). Assuming that mitochondria account for roughly 10% of the total cell volume (17), both DBP and mRNAs exist within the organelle at a concentration of about 1 mM. Since the K d of DBP for binding to RNA is 0.5 to 1 nM, it is predicted that practically all mitochondrial mRNAs exist in vivo in complex with DBP. We have argued previously that mRNAs not protected at their 39 ends by bound DBP are subject to rapid degradation by an NTP-dependent 39 exoribonuclease which is present in yeast mitochondria (3). If so, then the steady-state level of mitochondrial mRNAs will largely be determined by the concentration of active DBP in the organelle. Likewise, if binding of DBP to dodecamer sites within the multi-genic pre-mRNAs is required for their processing to mature mRNAs (1, 3, 4), then the flux of nascent transcripts into the pool of mature mRNAs may also be regulated by the amount of free DBP. Therefore, a yeast cell would be able to efficiently adjust mitochondrial gene expression to growth conditions by either regulation of DBP expression itself or by regulation of its ability to bind RNA, possibly through post-translation modification. Virtually all high affinity RNA binding proteins recognize both structure and sequence in an RNA to accomplish specificity (10, 13). Although the dodecamer sequence is nearly palindromic, our data do not indicate that RNA secondary structure is required for DBP binding. Compensatory sequence changes to the dodecamer site which were predicted to preserve secondary structure nevertheless abolished DBP binding to RNA. Furthermore, calculations based on thermodynamic parameters predict that the dodecamer site exists in solution as a linear molecule, not as stable hairpin (18), although it is possible that such structures could be stabilized upon DBP binding (19). It appears, therefore, that DBP is unique among site-specific RNA binding proteins in that it recognizes RNA primarily based on its sequence. Compared with other RNA binding proteins, the binding affinity of DBP for its target RNA is among the highest reported (less than 1 nM for DBP versus those ranging from 1 to 100 nM or more for most others (20 –25). The higher affinity of DBP binding is probably due to a greater number of amino acidnucleotide contacts within the DBP–RNA complex, as
suggested by the observation that a change in nearly any one of the nucleotides within the dodecamer sequence is sufficient to greatly reduce binding affinity. REFERENCES 1. Osinga, K. A., De Vries, E., Van der Horst, G., and Tabak, H. F. (1984) EMBO Journal 3, 829 – 834. 2. Hofmann, T. J., Min, J., and Zassenhaus, H. P. (1993) Yeast 9, 1319 –1330. 3. Min, J., and Zassenhaus, H. P. (1993) Molecular & Cellular Biology 13, 4167– 4173. 4. Butow, R. A., Zhu, H., Perlman, P., and Conrad-Webb, H. (1989) Genome 31, 757–760. 5. Zhu, H., Conrad-Webb, H., Liao, X. S., Perlman, P. S., and Butow, R. A. (1989) Molecular & Cellular Biology 9, 1507–1512. 6. Zhu, H., Macreadie, I. G., and Butow, R. A. (1987) Molecular & Cellular Biology 7, 2530 –2537. 7. Min, J., and Zassenhaus, H. P. (1993) Journal of Bacteriology 175, 6245– 6253. 8. Min, J. J., Heuertz, R. M., and Zassenhaus, H. P. (1993) Journal of Biological Chemistry 268, 7350 –7357. 9. Muller, W. E., Okamoto, T., Reuter, P., Ugarkovic, D., and Schroder, H. C. (1990) Journal of Biological Chemistry 265, 3803– 3808. 10. Draper, D. E. (1995) Annual Review of Biochemistry 64, 593– 620. 11. Dake, E., Hofmann, T. J., McIntire, S., Hudson, A., and Zassenhaus, H. P. (1988) Journal of Biological Chemistry 263, 7691– 7702. 12. Laemmli, U. K. (1970) Nature 227, 680 – 685. 13. Rould, M. A., Perona, J. J., and Steitz, T. A. (1991) Nature 352, 213–218. 14. Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) Journal of Biological Chemistry 268, 27363–27370. 15. Mueller, D. M., and Getz, G. S. (1986) Journal of Biological Chemistry 261, 11816 –11822. 16. Ferguson, K. A. (1964) Metabolism 13, 985–1002. 17. Stevens, B. (1981) The Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance (Strathern, J. N., Jones, E. W., and Broadh, J. R., Eds.), pp. 471–504, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 18. Jaeger, J. A., SantaLucia, J., Jr., and Tinoco, I., Jr. (1993) Annual Review of Biochemistry 62, 255–287. 19. Ribas de Pouplana, L., Auld, D. S., Kim, S., and Schimmel, P. (1996) Biochemistry 35, 8095– 8102. 20. Ikeda, M., Arai, K., and Masai, H. (1996) European Journal of Biochemistry 238, 38 – 47. 21. Ghisolfi-Nieto, L., Josep, G., Puvion-Dutilleul, F., Amalric, F., and Bouvet, P. (1996) Journal of Molecular Biology 260, 34 –53. 22. Burd, C. G., and Dreyfuss, G. (1994) EMBO Journal 13, 1197– 1204. 23. Ashley, C. T., Jr., Wilkinson, K. D., Reines, D., and Warren, S. T. (1993) Science 262, 563–566. 24. Wahle, E., Lusig, A., Jeno, P., and Maurer, P. (1993) Journal of Biological Chemistry 268, 2937–2945. 25. Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M., and Skinner, M. A. (1990) EMBO Journal 9, 4145– 4153.
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Purification and Characterization of an RNA Dodecamer Sequence Binding Protein from Mitochondria of Saccharomyces cerevisiae Huilin Li and H. Peter Zassenhaus 1 Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
Received July 8, 1999
Saccharomyces cerevisiae mitochondrial mRNAs terminate at their 3* ends with a conserved dodecamer sequence, 5*-AAUAA(U/C)AUUCUU-3*. We have identified a nuclear-encoded protein (DBP) which specifically binds to the dodecamer sequence and have purified it to apparent homogeneity by RNA affinity chromatography. DBP consists of a single polypeptide of 55 kDa and binds to its RNA substrate with a 1:1 stoichiometry. Scatchard analysis determines that K d is 0.93 nM for the canonical dodecamer sequence (5*AAUAAUAUUCUU-3*) and 0.46 nM for the only naturally occurring variant (5*-AAUAACAUUCUU-3*) unique to oli1 gene. Based on the studies using mutant oligonucleotides, DBP appears to recognize primarily the nucleotide sequence of an RNA rather than its potential secondary structure. © 1999 Academic Press
Previous work in our laboratory identified a protein from mitochondria of Saccharomyces cerevisiae that specifically binds to a highly conserved dodecamer sequence, 59-AAUAA(U/C)AUUCUU-39, which is found at the 39 ends of all mitochondrial mRNAs (1, 2). This sequence is encoded within multi-cistronic pre-mRNAs and appears to be a site for specific cleavage event that occurs a few nucleotides downstream of the dodecamer sequence (2). Since mitochondrial mRNAs in yeast are not polyadenylated, mature mRNAs terminate with the dodecamer sequence. We identified a protein (termed DBP for dodecamer binding protein) that bound to that sequence whether located internally within an RNA or at its 39 terminus (3). Although direct experimental evidence for the functions of DBP is not yet available, it is likely that it 1
To whom correspondence should be addressed. Fax: (314) 7733403. E-mail: [email protected]. Abbreviations used: DBP, dodecamer sequence binding protein; EMSA, electrophoretic mobility shift analysis; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
plays an important role in pre-mRNA processing and stabilization of mRNAs based upon the analysis of strains with mutant dodecamer sequences. Point mutations within the v intron dodecamer sequence eliminate endoribonucleolytic processing of v-containing RNAs at that site (4 – 6). RNA oligonucleotides containing the mutant v dodecamer sequence also show greatly reduced binding to DBP in vitro (3), suggesting that DBP plays a role in directing site-specific cleavage of that RNA. Deletion of the dodecamer site plus some flanking sequence associated with the var1 gene results in aberrantly processed var1 transcripts that appear to be more rapidly degraded than their wild-type counterpart (4). This observation together with the fact that most mitochondrial mRNAs terminate with a dodecamer sequence (2) suggests that DBP binding to mature mRNAs is important for their stability in vivo. In vitro, we have shown that DBP binding to the 39 region of an RNA protects it from digestion by an NTP-dependent 39 exoribonuclease that is found in yeast mitochondria (3). This enzyme appears to function in the turnover of mitochondrial RNAs (7, 8), and binding of DBP to the 39 ends of mitochondria mRNAs may therefore regulate mRNA stability by regulating the access of that enzyme to mRNAs. To further delineate the role of DBP in RNA processing and mRNA function, we have purified DBP and characterized its interaction with RNA substrates of differing sequence. Unlike most other sequencespecific RNA binding proteins (9, 10), DBP binding to its substrate does not appear to require secondary structure in the RNA that is based upon base-pairing. Rather, DBP appears to recognize the primary sequence directly in a single-stranded RNA. EXPERIMENTAL PROCEDURES Purification of DBP. A1237 m161 (Mata ad1 ura3 leu2 nuc1-1 : : LEU2) was grown to late log phase in YPGal (1% yeast extract, 2% Bacto peptone, 4% galactose) at 30°C with vigorous
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shaking. About 1.5 kg wet weight of cells was harvested from 60 liters of culture and mitochondria were prepared as described previously, except that they were not gradient purified (11). The mitochondrial preparation was suspended in 150 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 0.5 M KCl, 5 mM MgCl 2, 1 mM EDTA, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 0.1% Triton X-100) and sonicated on ice for 10 min. The suspension was clarified by centrifugation at 10,000 3 g for 20 min. The pellet was processed two more times as above (using 100 ml lysis buffer each time) and the supernatants were combined and centrifuged at 100,000 3 g for 1 h. The resulting supernatant (about 360 ml) was dialyzed overnight against Buffer A (10 mM Tris–HCl, pH 7.5, 5 mM MgCl 2, 0.1 mM EDTA, 2 mM DTT, 10% glycerol) plus 0.1 M KCl and applied to a heparin-Sepharose column equilibrated in Buffer A containing 0.15 M KCl. After washing with 5 column volumes of the same buffer, DBP was eluted with a 500 ml gradient of 0.15– 0.5 M KCl in Buffer A (Fig. 1A and Table 1). Fractions containing DBP binding activity were pooled and concentrated three fold by dialysis against 20% polyethylene glycol (MW 20,000) in Buffer A and the concentration of KCl adjusted to 0.2 M KCl. DBP binding activity was further purified by RNA affinity chromatography. A chimeric RNA/DNA oligonucleotide was synthesized so that the first 30 residues (from the 59 end) were poly(dA) which were then followed in the oligonucleotide by the dodecamer containing RNA sequence 59-AUAAAAUAACAUUCUUAAUU-39. The chimeric oligonucleotide (550 mg) was annealed to a poly(dT)-cellulose support (Type 7, Pharmacia; 500 mg) and equilibrated with Buffer A plus 0.2 M KCl. The binding capacity of this column was approximately 0.1 nMole of DBP. A portion of the dialysate (;20%) was applied to the RNA affinity column and, after washing with 10 column volumes of Buffer A containing 0.2 M KCl, DBP was eluted with a 50 ml linear gradient of KCl (0.2– 0.6 M in Buffer A). The amount of protein in each step of the purification was determined using the Pierce BCA Protein Assay Reagent and bovine serum albumin (BSA) as a standard. The amount of protein after the final step was estimated from Coomassie Brilliant Blue staining of SDS– PAGE fractionated samples in comparison to the staining intensity of known amounts of BSA. Preparation of different RNA substrates. RNA oligonucleotides were gel-purified for selection of full-length species and the amount of purified oligonucleotide quantified by measuring UV absorbence at 260 nm. The nomenclature and sequence of the oligonucleotides used here were: (1) Var, 59-AUAAAAUAAUAUUCUUAAUU-39, which contained the canonical dodecamer sequence as underlined plus the flanking 4 nucleotides on each side as found at the dodecamer site associated with the var1 gene; (2) a set of sequence variants, Var 3U3 C, Var 3,4UA3 AU, Var 6U3 C, Var 10C3 A, where the subscript denotes the nucleotide position within the dodecamer sequence that was altered followed by the sequence change; and (3) NS, 59-AUAUAUUAAUAUAUAUACUU-39, an oligonucleotide with the same sequence composition as Var which was used as a control for non-specific binding. Oligonucleotides were 59-end labeled with T4 polynucleotide kinase and [g- 32P]ATP (Amersham) according to instructions supplied by the enzyme’s manufacturer (New England Biolabs). Quantification of DBP binding activity by electrophoretic mobility shift assay (EMSA). DBP binding activity was measured in a 15 ml reaction mixture containing 10 mM Tris–HCl, pH 7.5, 200 mM KCl, 1 mM EDTA, 2 mM DTT, 0.1 mg/ml tRNA (Sigma), 10% glycerol, and 32 P-labeled oligonucleotide Var. The reaction mixture was incubated at 4°C for 15 min and then fractionated by PAGE as described (3). Radioactivity was visualized by scanning the gel with a Molecular Dynamics PhosphorImager. Quantitative measurement of DBP binding activity was performed using saturating amounts (10 nM) of oligonucleotide Var 6U3 C. The amount of DBP was calculated based on the 1:1 stoichiometry between DBP and RNA.
UV cross-linking. UV cross-linking was performed as previously described (3). For in situ UV cross-linking, the complex between a 32 P-labeled Var oligonucleotide and purified DBP was first fractionated on a native gel and then the gel piece containing the labeled complex was irradiated with 256 nm UV light for 10 min (12 joules). The irradiated complex was eluted from the crushed gel by soaking in Laemmli loading buffer (12) and fractionated by SDS–PAGE. Determination of dissociation constants (K d ) for the binding between DBP and RNA oligonucleotides. A series of binding reactions containing a fixed concentration of DBP (50 pM) were performed in which the concentration of the labeled RNA oligonucleotide varied from 0.05 nM to 50 nM. The amount of bound and free oligonucleotide was determined by EMSA. The equilibrium dissociation constant was calculated by Scatchard analysis. Glycerol gradient sedimentation. 100 ml of a reaction mixture containing 32P-labeled oligonucleotide Var and purified DBP was layered on top of a 4.0 ml 10 –35% (v/v) glycerol gradient in Buffer A containing 0.2 M KCl. The gradient was centrifuged at 40,000 rpm at 4°C for 21 h in a Beckman SW 50.1 rotor. After centrifugation, 100 ml fractions were collected from the bottom of the gradient and assayed for the DBP–RNA complex by mobility shift assay. Protein standards of known molecular weight were centrifuged in a parallel gradient. Gel filtration. Gel filtration of complexes between DBP and 32Plabeled RNA was performed on Sephacryl S-200 (0.8 3 30 cm) equilibrated in Buffer A containing 0.2 M KCl. Elution of DBP–RNA complexes was determined by EMSA. Blue Dextran was used for determination of the void volume (V o). Ferguson analysis. Reaction mixtures containing 32P-labeled RNA-DBP complexes were electrophoresed on 4%, 6%, 8% and 10% native polyacrylamide gels and the relative electrophoretic mobilities (Rm) measured relative to the dye front (bromophenol blue). Molecular weights were calculated from the Ferguson Plot as described (16).
RESULTS Purification of DBP. DBP was purified from a mitochondrial extract by heparin-agarose chromatography (Fig. 1, Panel A) followed by RNA affinity chromatography (Fig. 1, Panel B). The two sequential chromatographic steps purified DBP by about 10,000fold with a 30% yield (Table 1). Based upon the amount of DBP found in whole cell lysates, it was estimated that each yeast cell contains about 1000 to 2000 DBP molecules. When the peak fraction from the eluate of the RNA affinity column was analyzed by SDS–PAGE and silver staining, the major polypeptide observed had an apparent molecular weight of 55 kDa (Fig. 1, Panel C). Of the proteins eluting from the RNA affinity column, only p55 co-fractionated with DBP activity (data not shown). Previously, we had shown that UV irradiation of reaction mixtures containing a mitochondrial extract and 32P-labeled Var gave rise to three 32P-labeled polypeptide with apparent molecular weights of 70, 55, and 19 kDa (3). When the purified DBP preparation was similarly analyzed, we likewise observed the formation of those three cross-linked species (Fig. 1, Panel D, lane 1), although p55 was always the major crosslinked species. The formation of those species was inhibited by the inclusion of 100 fold excess unlabeled Var (Panel D, lane 2) but not by the non-specific RNA
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FIG. 1. Purification of DBP from yeast mitochondria. DBP was purified by sequential chromatography of a mitochondrial extract through heparin-Sepharose (Panel A) and an RNA affinity column (Panel B). Solid circles, relative amounts of DBP as determined by mobility shift assay and quantification of the bound complex by densitometry of the gel using the Molecular Dynamics PhosphorImager; open circles, concentration of KCl. Panel C, SDS–PAGE of a peak fraction from the eluate of the RNA affinity column. Lane 1, sample buffer blank; lane 2, a peak fraction from the RNA affinity chromatography. Only the p55 polypeptide co-fractionated with binding activity eluted from the RNA affinity column. The mobilities of molecular size markers (in kilodaltons) are indicated on the left of the gel. Panel D, UV crosslinking of p55 to RNA oligonucleotides. In lanes 1–3, purified DBP was incubated with RNA oligonucleotides, illuminated by UV light and then analyzed by SDS–PAGE. Lane 1, 32P-labeled Var only; lane 2, 32P-labeled Var plus 100-fold molar excess of unlabeled Var; lane 3, 32P-labeled Var plus 100-fold molar excess of NS. In lane 4, the complex between 32P-labeled Var and purified DBP was first electrophoretically fractionated on a native gel before exposure to UV light in situ. The UV-irradiated complex was eluted from the gel and then analyzed by SDS–PAGE.
oligonucleotide, NS (Panel D, lane 3). When DBP/RNA complexes were first fractionated by native gel electrophoresis before UV irradiation in situ, followed by SDS–PAGE of the eluted material, p55 was again the major cross-linked species observed (Panel D, lane 4) with only minor amounts of p70 and p19 detectable after long exposures of the gel. We interpret these results to indicate that p55 alone accounts for the dodecamer-site binding activity present in mitochondrial lysates.
towards the six different oligonucleotides mirrored the behaviour of the activity present in the mitochondrial extract (Fig. 2). As noted before (3), DBP binding to Var 6 U3 C was greater than towards the canonical dodecamer sequence (Var). In contrast, both the purified and crude activities showed much less binding to the sequence variants and none at all to the NS oligonucleotide. Although Fig. 2 shows a stronger binding of Var 3U3 C to purified DBP than to the activity in a crude mitochondrial lysate, a detailed analysis using
Binding specificity of the purified DBP. To determine whether the purified DBP had the same binding specificity as the activity previously described in mitochondrial extracts (3), a set of six 32P-labeled RNA oligonucleotides of differing sequences were synthesized for comparison of binding activities. Overall, we found that the binding activity of the purified DBP
TABLE 1
Purification of the Dodecamer-Site Binding Protein Step
Protein (mg)
DBP (nmole)
Yield (%)
Mitochondria Mitoplasts 100,000 3 g Supernatant Heparin-Sepharose RNA Affinity
9,000 5,000 3,500 100 ;0.1
— — 2.0 1.1 0.6
— — 100 53 30
FIG. 2. Binding specificity of the purified DBP compared to the activity present in a mitochondrial extract. A mitochondrial extract (lanes 1– 6) and the purified DBP (lanes 7–12) were incubated with a set of six 32P-labeled RNA oligonucleotides of differing sequence. The sequences of RNA oligonucleotides (indicated above each lane) used in the binding reactions are described in the “Experimental Procedures.” The binding reaction was carried out under standard conditions containing 1 nM oligonucleotides. The labeled complexes were analyzed by mobility shift assay and visualized by a Molecular Dynamics PhosphorImager.
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one associated with the oli1 gene (1, 2). Here, a U to C replacement occurs at position 6 of the canonical dodecamer sequence. To determine whether that sequence change affected DBP binding, we compared the binding affinity of DBP towards oligonucleotide Var vs Var 6U3 C. By a Scatchard analysis, the K d using Var 6U3 C was found to be 0.46 nM whereas with Var it was 0.93 nM (Fig. 3).
FIG. 3. Scatchard analysis for the measurement of DBP binding affinity to RNA oligonucleotides. A series of binding reactions containing a fixed concentration of DBP (50 pM) were performed in which the concentration of the labeled RNA oligonucleotides, Var and Var 6U3 C, varied from 0.05 nM to 50 nM. The bound and free oligonucleotides were fractionated by EMSA and quantified on a Molecular Dynamic PhosphorImager. The dissociation constants (K d) of DBP for Var or Var 6U3 C were determined by Scatchard analysis.
competition assays revealed that, as with crude lysate, purified DBP bound weakly to Var 3U3 C (data not shown). These results indicate that purified DBP accounts for the binding activity observed in mitochondrial lysates. Binding affinity. All dodecamer sites in the mitochondrial genome have the same sequence except the
Stoichiometry of DBP–RNA complex. To determine whether DBP bound RNA as a monomer or higherorder polymer, we measured the apparent size of the complex formed between purified DBP and a 20-base long RNA oligonucleotide. Three different techniques were employed so as to reduce the likelihood that the measured size was confounded by conformational artifacts. Whether by glycerol gradient sedimentation (Fig. 4, Panel 1), gel filtration (Fig. 4, Panel 2), or gel electrophoresis combined with a Ferguson analysis (Fig. 4, Panel 3), the observed size of the complex was consistently found to be between 50 – 60 kDa. Since the purified DBP itself has a molecular weight of about 55 kDa, these results suggest that DBP bind RNA as a monomer. We also determined that complexes contained only a single RNA molecule (data not shown), indicating that the stoichiometry of DBP binding to RNA was 1:1. The requirement of secondary structure in the RNA for binding activity. In many cases, the secondary structure of an RNA substrate plays an important role for site-specific binding (16, 17). The dodecamer se-
FIG. 4. Apparent size of the DBP–RNA complex. The apparent size of the complex between purified DBP and 32P-labeled Var was estimated by glycerol gradient sedimentation (Panel A), gel filtration (Panel B), and native gel electrophoresis in a Ferguson Plot analysis (Panel C). The standard marker proteins used in these experiments were b-AMS (b-amylase, 200 kDa), YAD (yeast alcohol dehydrogenase, 150 kDa), BSA (bovine serum albumin, 66 kDa), CA (carbonic anhydrase, 29 kDa) and cyt-C (cytochrome C, 12.5 kDa). DBP–RNA complexes were detected by electrophoresis on a native polyacrylamide gel. (Panel C) 32P-labeled DBP–RNA complexes were fractionated on 4%, 6%, 8% and 10% native polyacrylamide gels in parallel to the protein standards. Relative electrophoretic mobilities (Rm) were measured with respect to the dye front (bromophenol blue) and the Ferguson Plot was constructed according to Ferguson. The solid bars indicate the position of the DBP–RNA complex. 743
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FIG. 5. The role of potential secondary structure of the dodecamer sequence for DBP binding. The potential secondary structure of the canonical dodecamer sequence in the RNA oligonucleotide Var is diagrammed at the left. The dodecamer sequence resides within the shaded portion with each residue of the binding site numbered starting from the 59 end. The sequence alterations are illustrated at the middle of the diagram. Var 1,2 contained substitution of U’s for A’s at positions 1 and 2; In Var 11,12, A’s replaced U’s at positions 11 and 12; and Var 1,2/11,12 was synthesized so as to contain both sets of above sequence changes. 32P-labeled Var and the different modified versions were reacted separately with purified DBP in the standard binding reactions and analyzed by EMSA, which was then visualized by a Molecular Dynamics PhosphorImager.
quence is nearly palindromic (AAUAAU\AUUCUU). This feature may allow an oligonucleotide to form a stem-loop structure as illustrated in the left diagram of Fig. 5 for oligonucleotide Var. The dodecamer sequence is highlighted within the shaded box. Residues within the binding site are numbered starting from the 59 end. To test whether secondary structure is required for DBP binding, oligonucleotides were synthesized that contained sequence changes which were predicted to disrupt potential base pairing. An oligonucleotide was synthesized wherein the two adenosine residues at positions 1 and 2 were replaced by uridines; likewise, another oligonucleotide was made replacing the two uridine residues at positions 11 and 12 with adenosine residues (Fig. 5). A third oligonucleotide was also made containing both sets of the above sequence changes so that potential base pairing would be restored because of compensatory base substitutions. If secondary structure was required for DBP binding, we predicted that this last oligonucleotide would show binding activity whereas the first two would not. As shown in Fig. 5, Panel B, DBP failed to bind to any of these modified oligonucleotides, suggesting that secondary structure within the dodecamer site is not necessary for binding activity. DISCUSSION Several lines of evidence indicated that p55 was responsible for the DBP activity detected in a mitochondrial lysate. First, purified p55 shared the same binding specificity as the DBP activity in a crude lysate. Second, p55 was the only polypeptide to co-fractionate with DBP activity by RNA-affinity chromatography. Third, both p55 and crude DBP gave a similar spec-
trum of labeled adducts when UV cross-linked to a 32 P-labeled RNA oligonucleotide. In each case, the major adduct had an apparent molecular weight of about 55 kDa, similar to that of purified p55. The origin of the minor adducts of approximately 19 and 70 kDa is unclear at the moment. Finally, the native size of the binding complex was between 50 – 60 kDa, again similar to that of purified p55. Understanding the basis for the specific interaction between DBP and its RNA substrate requires knowing the stoichiometry of the complex. Many site-specific DNA binding proteins bind to their substrate as dimers when the DNA sequence within the binding site itself is symmetric. To determine the number of DBP molecules in a complex with its RNA substrate, the size of the complex was measured by three independent experimental approaches. All three (glycerol gradient sedimentation, gel filtration, and native gel electrophoresis) gave the same result: the size of the native complex was between 50 and 60 kDa, similar to the apparent molecular weight (55 kDa) of DBP. From these results we conclude that DBP binds to its RNA substrate as a monomer. Furthermore, from a determination of the number of RNA molecules in a single complex with DBP, it appears that one DBP molecule binds to one RNA substrate molecule. When present in mitochondrial RNAs, the dodecamer site is not tandemly duplicated. Given a 1:1 stoichiometry between DBP and RNA in a complex, it appears that DBP binding in vivo to either pre-mRNAs or mature mRNAs would not exhibit cooperativity. Consequently, the regulation of DBP binding to premRNAs and mRNAs may rely on either mass action, which would demand large changes in the concentration of DBP to change the relative amount of RNA in
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complex with DBP, or possibly on post-translational modification, such as phosphorylation. An increasing number of RNA binding proteins have been shown to regulate their binding affinity via phosphorylation (14). We have demonstrated that treatment of DBP with alkaline phosphatase abolishes specific, high affinity RNA binding (manuscript in preparation), suggesting that reversible phosphorylation may play a role in the regulation of DBP binding activity. Each yeast cell contains between 1000 to 2000 DBP molecules, which is close to the number of mitochondrial mRNA molecules present within the organelle (15). Assuming that mitochondria account for roughly 10% of the total cell volume (17), both DBP and mRNAs exist within the organelle at a concentration of about 1 mM. Since the K d of DBP for binding to RNA is 0.5 to 1 nM, it is predicted that practically all mitochondrial mRNAs exist in vivo in complex with DBP. We have argued previously that mRNAs not protected at their 39 ends by bound DBP are subject to rapid degradation by an NTP-dependent 39 exoribonuclease which is present in yeast mitochondria (3). If so, then the steady-state level of mitochondrial mRNAs will largely be determined by the concentration of active DBP in the organelle. Likewise, if binding of DBP to dodecamer sites within the multi-genic pre-mRNAs is required for their processing to mature mRNAs (1, 3, 4), then the flux of nascent transcripts into the pool of mature mRNAs may also be regulated by the amount of free DBP. Therefore, a yeast cell would be able to efficiently adjust mitochondrial gene expression to growth conditions by either regulation of DBP expression itself or by regulation of its ability to bind RNA, possibly through post-translation modification. Virtually all high affinity RNA binding proteins recognize both structure and sequence in an RNA to accomplish specificity (10, 13). Although the dodecamer sequence is nearly palindromic, our data do not indicate that RNA secondary structure is required for DBP binding. Compensatory sequence changes to the dodecamer site which were predicted to preserve secondary structure nevertheless abolished DBP binding to RNA. Furthermore, calculations based on thermodynamic parameters predict that the dodecamer site exists in solution as a linear molecule, not as stable hairpin (18), although it is possible that such structures could be stabilized upon DBP binding (19). It appears, therefore, that DBP is unique among site-specific RNA binding proteins in that it recognizes RNA primarily based on its sequence. Compared with other RNA binding proteins, the binding affinity of DBP for its target RNA is among the highest reported (less than 1 nM for DBP versus those ranging from 1 to 100 nM or more for most others (20 –25). The higher affinity of DBP binding is probably due to a greater number of amino acidnucleotide contacts within the DBP–RNA complex, as
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