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J. Biol. Chem., Vol. 275, Issue 40, 31183-31190, October 6, 2000

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Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate^*

Tatyana Naryshkina, Dragana Rogulja^§¶, Larisa Golub^§, and Konstantin Severinov^**

From the Waksman Institute for Microbiology and the  Department of Genetics, Rutgers, State University of New Jersey, Piscataway, New Jersey 08854

Received for publication, May 8, 2000, and in revised form, July 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP)  and  subunits during the formation of ₂, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing  conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with . The -interacting domain is held together by protein-protein interactions between  segments F and I. Residues in catalytically important  segments H and I directly participate in  binding; substitutions of strictly conserved segment H Asp¹⁰⁸⁴ and segment I Gly¹²¹⁵ abolish ₂ formation in vitro and are lethal in vivo. The importance of these  amino acids in  binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of  Asp¹⁰⁸⁴ in A135, the -like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest -like subunit. However, the A135-AC40 interaction is weak compared with the E. coli - interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cellular RNA polymerases (RNAPs)¹ are large, multisubunit enzymes. A typical prokaryotic RNAP core contains 5 polypeptides with a total molecular mass of ~400 kDa. Core RNAP from eukaryotes and archaea contain 10-14 subunits with a total molecular mass in excess of 500 kDa. Sequence alignments of RNAP subunits reveal extensive similarities; each of the two largest RNAP subunits, which are the most evolutionarily conserved, contains 8-9 colinear segments with many invariant amino acids (1, 2). RNAPs from different sources are also homologous structurally; low resolution (16-35 Å) three-dimensional models of Escherichia coli, and RNAP II and RNAP I from yeast obtained by means of electron crystallography reveal significant similarities (3-5).

Evolutionarily conserved subunit segments probably form distinct functional domains common to all RNAPs. Genetic data support this notion; mutational changes of conserved residues selectively destroy distinct partial functions of the enzyme (e.g. the transition from abortive initiation to productive elongation (Ref. 6), or phosphodiester bond synthesis (Ref. 7)), but leave other functions unperturbed. However, isolated subunits themselves do not possess any of the partial functions of the whole enzyme (8). Therefore, RNAP functional sites are either formed allosterically upon the enzyme assembly, or are located at subunit interfaces. Thus, understanding inter- and intrasubunit interactions should provide insights in RNAP mechanism and regulation.

E. coli RNAP assembles in vivo and in vitro according to the following scheme: -₂-₂-₂' (9). The ₂ assembly intermediate appears to be evolutionary conserved, and an ₂-like RNAP II subassembly was isolated from yeasts (10, 11). Further, mutations in E. coli , and in its yeast RNAP II counterpart, Rpb3, that affect the ₂ formation in their respective systems occur in homologous positions (12, 13).

The  and ' homologues are naturally fragmented in some archaea and cyanobacteria (14, 15). The assembly pathway should be more complex in organisms with split , ' homologues, but this has not been investigated. Our own work with E. coli defined four separable domains in  and three in ' (16, 17), that correspond to natural fragmentation sites in some of the archaea and cyanobacteria. The ability to generate functional E. coli RNAP using subunit domains greatly aided the study of RNAP assembly and biochemical functions of assembly intermediates. A combination of fragmented RNAP reconstitution, limited proteolysis, and protein-protein co-immobilization assays was used to demonstrate that determinants of specific  binding reside in the C-terminal assembly-competent structural module of , containing amino acids 907-1342 (conserved segments H and I; Ref. 18). However, similar analysis performed by the Ishihama group (19) suggested that the primary determinants of  binding include conserved segments F and G, corresponding to E. coli  amino acids 800-900.

The discrepancy between the two sets of data illustrates problems associated with in vitro coimmobilization assays, which can be artifact-prone. Thus, alternative approaches are needed to characterize the - interaction in molecular detail. Here, we used the method of yeast two-hybrid analysis to analyze the interaction of the  and  subunits during ₂ formation. Our results support the conclusion of Wang et al. (18) and directly implicate two strictly conserved amino acids in  segments H and I in interactions with .

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteins and Plasmids-- Gal4-based two-hybrid plasmids pPC97 (bait plasmid) and pPC86 (prey plasmid) were described in Ref. 20.

Derivatives-- The two-hybrid plasmids expressing  NTD (rpoA codons 1-235) were constructed by PCR cloning using E. coli expression plasmids containing either wild-type rpoA or rpoA harboring the R45A mutation as templates (18). E. coli plasmid expressing His₆-NTD, and conditions for protein expression and purification are described in Ref. 21.

Derivatives-- The two-hybrid plasmids expressing various fragments of  used in this work were constructed by PCR cloning using E. coli pMKSe2 expression plasmid (22) as a template. Site-specific PCR mutagenesis and standard recloning steps were used to introduce point mutations in yeast two-hybrid of E. coli expression plasmids. Full-length  and ' used in in vitro RNAP assembly studies were obtained from expression plasmids pMKSe2 and pT7' (23), respectively. Overexpression was induced with 1 mM isopropyl--D-thiogalactoside at 30 °C for 3 h. The proteins in inclusion bodies were then prepared according to Ref. 21.

Yeast RNAP Subunit Derivatives-- The two-hybrid plasmids expressing various fragments of yeast RNAP subunits were constructed using appropriate primers by high fidelity PCR with yeast genomic DNA as a template. Plasmid pNOY302, which harbors the entire A135 gene, was provided by M. Nomura and is described in Ref. 24.

Yeast Strains and Techniques-- Yeast were transformed using standard lithium acetate procedure, and transformants were selected on appropriate drop-out media. For two-hybrid assay, we used the LNY 327 tester strain provided by L. Neigeborn. This strain is identical to the PCY2 strain of Chevrai and Nathans (20). The interaction between two-hybrid constructs was scored using filter assays, or quantitative colorimetric assay using ortho-nitrophenyl--galactoside as a substrate (25). The following formula was used to calculate -galactosidase production: -galactosidase activity (Miller units) = 10,000 × (OD₄₂₀  1.75 × OD₅₅₀)/(OD₆₀₀ × t), where OD₄₂₀, OD₅₅₀, and OD₆₆₀ are optical density of the reaction at 420, 550, and 600 nm, respectively at time t (min) of the reaction.

Yeast tester strain NOY302-1a was provided by M. Nomura and has been described previously (26).

Reconstitution of ₂ Complexes and RNAP-- Purified His₆ NTD was mixed with  or its derivatives (from washed inclusion bodies) at a molar ratio of 1:1 and a total protein concentration of not more than 0.5 mg/ml in reconstitution buffer (40 mM Tris-HCl, pH 7.9, 100 mM NaCl, 10 mM EDTA, 20 mM MgCl₂, 10 µM ZnCl₂, 20% (v/v) glycerol, 2 mM -mercaptoethanol, 6 M guanidine HCl) and dialyzed overnight at 4 °C against 2 × 2 liters of the same buffer without guanidine HCl. For RNAP reconstitution, proteins were combined at a molar ratio ::' of 1:1:2. At these conditions, unassembled ' precipitated during dialysis and was removed by low speed centifugation.

Ni²⁺-NTA-Agarose Co-immobilization Binding Assays-- Protein complexes (about 10 µg) were mixed with pre-equilibrated Ni²⁺-NTA-agarose beads (Qiagen) in 50 µl of reconstitution buffer and incubated for 10 min at room temperature with gentle mixing. The beads were pelleted by centrifugation and washed twice with 500 µl of buffer with 10 mM imidazole. The protein samples were then eluted from the beads with buffer containing 100 mM imidazole and analyzed by SDS-PAGE.

In Vitro Transcription-- 10 µl of RNAP reconstitution reaction was incubated for 15 min at 37 °C in the presence of 1 µg of recombinant ⁷⁰ subunit (23), and 50 ng of the T7A1 promoter-containing DNA fragment (22), 0.5 mM CpA, with or without 25 µg/ml rifampicin. Reactions were initiated by the addition of NTP (final concentration 25 µM ATP, CTP, and GTP, 1 µM [-³²P]UTP (1000 Ci/mmol). Reactions proceeded for 15 min at 37 °C, and were terminated by the addition of urea containing loading buffer. Reaction products were resolved by denaturing 20% PAGE and revealed by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Interaction between E. coli RNAP  and  Can Be Detected in Yeast Two-hybrid System-- For our research, it was critical to establish that the interaction between  and the dimeric  subunit could be studied by two-hybrid approach, and that mutations that decrease this strong interaction can be detected in yeast. As an initial test, we used the N-terminal domain of  (NTD, amino acids 1-235) and the  subunit fragment containing amino acids 711-1246, which specifically interact with each other in vitro (18). -NTD was fused to the GAL4 DNA binding domain (Gal4-DB) of the bait plasmid pPC97, and _711-1246 was fused to the GAL4 activation domain (Gal4-AD) of the prey plasmid pPC86 (20). High levels of -galactosidase were observed when both plasmids were present (intense blue color developed in less than 30 min in a standard filter assay; 440 Miller units of -galactosidase activity in a quantitative liquid assay, see "Materials and Methods"); no activity was observed when either fusion plasmid was used separately, as expected (cells remained white after overnight incubation on a filter, less than 1 Miller unit of -galactosidase activity). Importantly, when pPC97_1-235, carrying a mutation that changes  Arg⁴⁵ for Ala and weakens the interaction with  (12), was used, no activity was detected even in the presence of pPC86_711-1246 (white color, less than 1 Miller unit of -galactosidase activity, data not shown). Thus, we can detect the - interaction and amino acid changes that affect this interaction using the two-hybrid system. This result sets the stage for more detailed analyses of RNAP assembly described below.

Defining the Minimal Fragment of  Capable of Interaction with -- Our starting  two-hybrid construct, pPC86_711-1246, contained 535 rpoB codons and corresponded exactly to the smallest  fragment that specifically interacted with  in vitro (18). The _711-1246 fragment spans four universally conserved  segments, F, G, H, and part of I. To determine the smallest  fragment capable of interacting with , we engineered pPC86_711-1246 derivatives with deletions at either the beginning or the end of the  moiety, and tested these new plasmids for their ability to elicit -galactosidase production in yeast cells harboring pPC97_1-235. The results are schematically presented in Fig. 1.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Determining the minimal  fragment capable of interacting with  in the two-hybrid screen. The horizontal structure at the top represents the primary sequence of . Evolutionarily conserved regions are shaded gray and labeled E-I according to Ref. 2. The open boxes represent regions containing large deletions found in  homologues from Gram-positive bacteria and chloroplasts. Dispensable regions are represented as bars above the primary structure. The locations of Lys1065 and His1237, which cross-link to initiating nucleotide analogs (27), are indicated (). Below the primary structure are the results of mapping the N and C termini of Gal4-DB-NTD-interacting Gal4-AD- fragments using two-hybrid assay, starting with the 711-1246 fragment characterized by Wang et al. (Ref. 18). The results of the standard -galactosidase plate assay are presented as either "+" or ""; the numerical values to the right represent the results of liquid -galactosidase assay in Miller units. All  fragment hybrids generated less than 1 Miller unit -galactosidase activity when combined with control R45A-Gal4-DB hybrid (data not shown).

As can be seen, _800-1246 hybrid interacted with the  hybrid. However, further deletion in segment F, _812-1246, abolished the interaction. At the C-terminal side, removal of  amino acids 1231-1246 had no effect on interaction with . However, the removal of additional 6  amino acids (1226-1231) abolished the interaction. The results suggest that  amino acids 711-800, which contain 80 non-conserved amino acids, and 10 amino acids from conserved segment F, and  amino acids 1231-1246, which contain most of  conserved segment I, including residue His¹²³⁷, which forms the 5'-face of the catalytic center (27), are not necessary for specific interaction with . We note, however, that these regions of  may still contribute to the overall strength of the binding, since the two-hybrid interaction may be saturated and insensitive to minor changes in the strength of binding.

The two-hybrid construct containing  amino acids 800-1231 was the smallest construct that interacted with  strongly (520 Miller units) and specifically (0.5 Miller units when used with ^R45A two-hybrid construct) (Fig. 1). Shorter constructs, constructed by site-directed mutagenesis, _812-1231, _832-1231, and _8oo-1225, failed to interact with  hybrid (<1 Miller unit). We also attempted to further narrow the interacting domain by performing nested Bal31 deletion mutagenesis at either the 5' or the 3' ends of the rpoB portion of pPC86_800-1231, transforming mutated plasmids in yeast cells harboring pPC97, and selecting interacting (Lac⁺) colonies. The DNA sequence of several interacting  plasmids was determined, and in all cases Bal31 deletions extended into the vector part of pPC86_800-1231, while rpoB codons 800 and 1231 were retained (data not shown). We conclude that _800-1231 is the minimal  fragment capable of  binding.

The minimal interacting fragment of , _800-1231, contains dispensable region II ( amino acids 907-1050) that is missing in some bacterial  subunits and in homologues from eukaryotes and archaea (28). This region tolerates an artificial split at around position 950 (16), and is highly susceptible to trypsin attack at position 907 and 911 (28). Thus, dispensable region II likely demarcates two structurally independent  modules. Earlier studies indicated that assembly-competent  module, _950-1342, which contains conserved segment H and I, interacted with His₆--NTD (18). However, _950-1342 binding was weak compared with the binding of the larger, _711-1342, fragment or full-sized . Moreover, _950-1342 interacted with His₆-^WT and His₆-^R45A in vitro with the same (low) efficiency (18). On the other hand, Nomura et al. (19) mapped the primary -binding site to  amino acids 737-904. We constructed pPC86-based two-hybrid plasmids with _800-911 (conserved segments F and G), and _907-1231, (conserved segments H and a small portion of I) and tested them for their ability to interact with . Neither pPC86_711-911 plasmid, nor pPC86_907-1231 elicited -galactosidase activity with pPC97_1-235 suggesting that  segments F and G, or segment H and I, alone do not interact with  in our assay (data not shown).

Our results suggest that  amino acids 801-811 and 1226-1231 in conserved regions F and I, respectively, contribute, either directly or indirectly, to the interaction with the  dimer. In the former scenario, both segment F and segment I residues interact with  to result in strong binding; neither segment binds  strongly enough for the interaction to be detected by two-hybrid assay. The latter scenario may involve an intrasubunit interaction between the two regions of , one containing segment F and G, and another containing segments H and I. One can envision, then, that the interaction of the two  fragments results in the formation of the structure which is fully capable of  binding. Two-hybrid constructs containing _800-911 and _907-1231 strongly interacted with each other (Fig. 2). In contrast, construct containing _812-911 did not interact with _907-1231. Likewise, no interaction was detected between two-hybrid constructs containing _800-911 and _907-1225. Thus, the residues required for  binding are also required for intrasubunit interactions between two  modules, consistent with the idea that segment F and I amino acids contribute to  binding indirectly.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   A strong intrasubunit interaction between  segments F and I. The structure at the top represents assembly-competent E. coli  subunit fragment corresponding to archaeal ' subunit (17). The Gal4-AD hybrids containing the fragments of  shown at the left were tested against the -Gal4-DB  fragments shown at the right, and the results are shown at far right.

Point Mutations in  Segment H and I Affect  Binding in Vivo and in Vitro-- In , substitution of the strictly conserved Arg⁴⁵ interferes with the  binding (12). We hypothesized that Arg⁴⁵ interacts with a negatively charged, conserved amino acid in , and that this interaction is responsible for the ₂ formation. Accordingly, we inspected the E. coli  fragment between amino acids 800 and 1231 to see if such amino acids could be found. Segment G contains no conserved negatively charged amino acids; segment F contains one such residue, Glu⁸¹³. This position was previously mutated to an Arg (29); the resulting RNAP had a catalytic defect but had no assembly defect, and therefore we did not study this position further. As can be seen from the alignment presented in Fig. 3A, segment H contains 4 negatively charged amino acids, Asp¹⁰⁶⁴, Asp¹⁰⁸⁴, Asp¹⁰⁹⁵, and Glu¹¹¹⁴, which are strictly conserved and thus fulfill our criteria for potential candidates involved in  binding. Residue Asp¹⁰⁶⁴ was mutated previously in the course of a mutational study of the RNAP initiating site (30). The results of that study revealed that  harboring the D1064A mutation was assembly-proficient, but the resulting RNAP was catalytically defective. Based on these results, position 1064 was excluded from our analysis. Segment I contains no conserved negatively charged amino acids. However, there are three Gly residues, at positions 1215, 1218, and 1228, which are strictly conserved. Previously, we had demonstrated that substitutions of evolutionarily conserved glycines for aspartic residues in the ' subunit result in assembly defects (31). Therefore, in addition to the three site-specific mutations changing two aspartates and one glutamate from segment H for alanines, we also substituted each of the three segment I glycines for aspartates. The mutations were engineered in pPC86_800-1231 background, and their effect on  binding in two hybrid system was studied. The results are shown above the alignment on Fig. 3A. Of the six mutations tested, only two, D1084A and G1215D, completely abolished the interaction as judged by both the filter and the liquid -galactosidase assays. G1218D showed weak, but clearly positive, interaction. The remaining mutations had no effect on  binding.

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Point mutations in  segment H and I interfere with interactions with . A, alignment of amino acid sequences of E. coli  segment H (top) and an N-terminal portion of segment I (bottom) and homologues from tobacco chloroplasts (Tobac.), archaeon Sulfolobus acidocaldarius (Su. ac.), three nuclear RNAPs from yeast (YP1, YP2, and YP3), and African swine fever virus (ASFV). Strictly conserved residues are indicated by bold typeface. The positions of the 5'-nucleotide cross-link sites and of site-specific mutations constructed in this work are indicated above the E. coli sequence. The values shown in parentheses represent the results of liquid -galactosidase assay (in Miller units) obtained with 800-1231-Gal4-AD hybrid carrying the site-specific mutations and the -NTD-Gal4-DB hybrid. B, segment H and I mutations were recloned into E. coli rpoB overproducing plasmid harboring a dominant RifR mutation. Mutant  subunits were overproduced, purified, and combined with hexahistidine-tagged NTD at conditions favoring RNAP assembly. Reactions were loaded onto Ni2+-NTA-agarose beads (L) and the unbound protein removed (F). The beads were then washed with buffer containing 25 mM imidazole (W), and then the bound proteins were eluted with buffer containing 100 mM imidazole (E). The protein fractions were then analyzed by SDS-PAGE on a 10% gel. C, experiment was performed as in B in the presence of the recombinant ' subunit. Reactions were analyzed by SDS-PAGE on a 10% gel (top) or a 6% gel (bottom), to separate the  and ' subunits. D, RNAP was assembled as in C; reactions were provided with purified 70 subunit, and the T7 A1 promoter-containing DNA fragment, in the presence, or in the absence of 25 µg/ml rifampicin. Transcription was initiated by the addition of CpA primer, ATP, CTP, GTP, and [-32P]UTP. Reaction products were resolved by denaturing 20% PAGE and visualized by autoradiography.

To confirm the  binding defects directly, we recloned all mutations into the pMKSe2(S531F) -overproducing plasmid (22). The  subunit expressed from pMKSe2(S531F) contains a rifampicin (Rif) resistance mutation changing Ser⁵³¹ to Phe. Therefore, Rif-sensitive host cells transformed with pMKSe2(S531F) grow on medium containing Rif. In contrast, cells transformed with pMKSe2(S531F) harboring the engineered segment H and I mutations did not form colonies in the presence of Rif, but were unaffected for growth in its absence. Thus, the rpoB mutations behaved as recessive lethals, a phenotype expected of assembly mutations.

The binding defect of  mutants was also showed directly, by studying their ability to co-immobilize on Ni²⁺-NTA-agarose through protein-protein interactions with hexahistidine-tagged NTD (Fig. 3B). In this experiment, the wild-type or mutant  subunits were prepared from inclusion bodies and combined with recombinant NTD in the presence of high concentration of denaturing agent guanidine HCl. The denaturant was dialyzed away at conditions favoring RNAP assembly, and reactions were allowed to bind to Ni²⁺-NTA-agarose beads. The beads were washed, and the bound protein was eluted with a buffer containing high concentration of imidazole. As can be seen, the wild-type  was efficiently immobilized on the sorbent in the presence of tagged NTD (Fig. 3B, lane 4), as expected. In contrast, most of  harboring the D1084A and G1215D substitutions was found in the flow-through (lanes 6 and 10) and did not associate with NTD (lanes 8 and 12). G1218D also caused a defect in NTD binding, but, in agreement with the two-hybrid results, the extent of this defect was weaker than those of either D1084A or G1215D (compare lane 20 with lanes 8 and 12). Similar analysis revealed that in agreement with the two-hybrid result substitutions at positions 1095, 1114, and 1228 had no effect in the co-immobilization assay (Fig. 3B, lane 24; and data not shown). We conclude that  positions 1084, 1215, and, to a lesser degree, 1218 are involved in  binding, and that the lethal in vivo phenotypes exhibited by the rpoB genes harboring substitutions in these positions are likely explained by the defect in the ₂ formation. In contrast, the lethal phenotype caused by substitutions at rpoB positions 1095, 1114, and 1228 must be due to alterations of some other essential RNAP function(s).

In principle, two classes of mutations can abolish protein-protein interaction. The mutations of the first class distort the native structure, perhaps through an allosteric mechanism, and thus prevent the interaction. The second, most interesting class, is true interaction mutations, which affect favorable interactions that bring the two proteins together. We investigated the ability of the  subunit harboring the D1084A and G1215D mutations to assemble into active RNAP in vitro in the presence of the wild-type ' and hexahistidine-tagged NTD. The assembly reactions were examined by Ni²⁺-NTA coimmobilization (Fig. 3C). The addition of ' corrected the NTD-binding defect caused by the D1084A mutation, and RNAP core enzyme was formed in good yield (Fig. 3C, lane 8). A steady-state in vitro transcription experiment revealed transcription activity in the RNAP assembly reactions containing  with D1084A substitution (Fig. 3D, lane 3). This transcription was rifampicin-resistant (lane 4), proving that the activity was due to the mutant RNAP, which also carries the S531F Rif resistance mutation, and not due to contaminating wild-type RNAP from the host, which is rifampicin-sensitive (see lanes 5 and 6). This result implies that the D1084A mutation does not grossly distort  structure and may be directly involved in interactions with . In contrast, no assembled RNAP was detected in the in vitro RNAP assembly reaction containing His-tagged , ', and  carrying the G1215D mutation (Fig. 3C, lane 12), and there was no transcription activity in G1215D assembly reactions (Fig. 3D, lanes 7 and 8). Thus, Gly¹²¹⁵ appears to play a structural role, and its involvement in  binding may be indirect.

Evolutionary Conservation of the - and -like RNAP Subunit Interactions-- In yeast, two -like subunits, AC40 and AC19, form a heterodimer that is functionally equivalent to the ₂ homodimer in prokaryotes (32). Interestingly, AC40 and AC19 are shared by RNAP I and RNAP III. Thus, interaction of the RNAP I -like subunit, A135, and its RNAP III homolog, C128, with the AC40/AC19 heterodimer could determine the relative amount of RNAP I and RNAP III in the cell. We wanted to know (i) whether C-terminal A135 and C128 fragments contained determinants for interaction with the -like subunits, as would be expected from our E. coli results, and (ii) whether C128 and A135 interact with the same -like subunit in the heterodimer. Two-hybrid plasmids expressing the N- and C-terminal halves of A135 and C128 as defined by the archaeal split were constructed. The two-hybrid plasmids were then tested for their ability to elicit -galactosidase production with complementary plasmids expressing either AC19 or AC40 hybrids. As control, we used the AC40 and AC19 pair, which had previously been shown to interact in two-hybrid assay. The results can be summarized as follows. First, as expected, AC40 fused to Gal4-DB showed strong positive interaction with AC19-Gal4-AD (120 Miller units). Second, the N-terminal fragments of the -like subunits, containing conserved segments A, B, C, D, and E (residues 1-728 and 1-717 in C128 and A135, respectively) did not interact with the -like subunits. Third, C-terminal fragment of C128 (residues 729-1149 conserved segments F, G, H, and I) also did not interact with either AC19 or AC40 hybrids, and thus no conclusions about the formation of RNAP III ₂-like structure could be made. Finally, weak (13 Miller units) interaction between the C-terminal fragment of A135 (residues 717-1203) with the AC19 hybrid, but not with the AC40 hybrid was detected.

Since the -like subunits AC19 and AC40 heterdimerize with high efficiency (32, see also above), the observed interaction between the A135_FGHI and AC19 two-hybrid constructs is likely due to A135_FGHI-Gal4DB interaction with AC40/AC19-Gal-AD heterodimer. We constructed an AC19 two-hybrid plasmid, which contained a double-alanine substitution at AC19 positions 78, and 79, corresponding to E. coli  positions 44 and 45, and should have abolished A135 binding to AC19. The resulting construct was unaffected in A135_FGHI interaction (15 Miller units), suggesting that conserved residues in the -motif of AC19 are not involved in A135 binding, or A135 interacts with AC40. To establish the specificity of two-hybrid interaction between AC40/AC19-Gal-AD and A135_FGHI-Gal4DB, we engineered a two-hybrid construct that expressed the C-terminal fragment of A135 with D935A substitution, which corresponds to E. coli rpoB^D1084A (Fig. 3A). No -galactosidase was produced when the AC19 and A135^D935A hybrids were combined in the same yeast cell (1 Miller unit). We conclude that (i) the C-terminal portion of A135 interacts with the largest -like subunit, AC40, and not with AC19, and (ii) A135 Asp⁹³⁵ is required for this interaction.

In E. coli, rpoB mutations that abolish the strong - interaction behave as haploinviable (see above). To test the functional consequence of D935A substitution in A135, which abolishes the A135-AC19/AC40 interaction in the two hybrid assay, yeast plasmid expressing the mutant A135 gene was constructed. The phenotype of this gene was tested using haploid NOY408-1a yeast tester strain (26). In this strain the chromosomal copy of A135 has been deleted, and the cells are kept viable because of the presence of a multicopy plasmid harboring an rDNA repeat under the control of GAL7 (RNAP II) promoter. Therefore, NOY408-1a is only viable when galactose is present and does not grow in the presence of glucose, when the GAL7 promoter is repressed. However, when a plasmid expressing a functional copy of A135 is introduced, NOY408-1a can grow in the presence of glucose, since rDNA is now transcribed by RNAP I. As can be seen, plasmid-borne A135^D935A, allowed robust growth on glucose (Fig. 4). Additional experiments established that the A135^D935A plasmid allowed growth on glucose at low temperature of 16 °C, and at high temperature of 37 °C (data not shown). We conclude that the A135^D935A gene is functional.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Yeast A135 harboring mutation that abolishes A135 interaction with AC40/AC19 in two-hybrid system is functional. Yeast tester strain NOY308-1a, which lacks A135 and is kept viable by the presence of Gal7-rDNA plasmid (26), was transformed with vector plasmid or plasmids expressing wild-type A135, or mutant A135 with D935A substitution. Transformants were spotted on rich medium containing galactose (top row) or glucose (bottom row) as carbon source. The results of growth after a 62-h incubation at 30 °C are presented.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The impetus for this study came from the apparently contradictory results of Nomura et al. (19) and Wang et al. (18), who localized the -binding determinants into non-overlapping fragments of E. coli . Nomura et al. claimed that  conserved segments F and G, harbor the primary -binding site; Wang et al. localized the binding site to  segments H and I; however, four conserved regions, F, G, H, and I, were required for strong and specific  binding. The results of our work are consistent with Wang et al., and demonstrate that  amino acids 1084 (conserved segment H) and 1215 (conserved segment I) are involved in  binding. We also demonstrate that there exists a strong intrasubunit interaction between  segments F and I, and that this interaction appears to be necessary for  binding. Finally, we present evidence that, in yeast, the second largest, -like RNAP I subunit A135 interacts with the larger -like subunit, AC40, and, unexpectedly, that the A135 mutation that abolishes this interaction is viable. While this research was in progress, a 3.3-Å structure of RNAP core from Thermus aquaticus (Taq) appeared (33). In addition, a medium resolution structure of yeast RNAP II also became available (34). Below, we interpret our findings in the context of the structural model of the Taq enzyme.

Intrasubunit Interaction of between Segment F and I Residues Is Important for  Binding-- Our results demonstrate that the smallest fragment of E. coli  capable of specific  binding spans amino acids 800-1231. The structural context of the corresponding fragment of Taq  is shown in Fig. 5 (A-C). As can be seen, the fragment has an elongated shape and consists of two domains. One domain is formed entirely by conserved segment G and contains the so-called "flexible flap" element, which is thought to interact with RNA at hairpin-induced pause sites (35), and harbors the site of trypsin attack (28). The second domain contains the entire conserved segment H as well as segments F_C and I_N, which are far away from each other in the primary sequence. The second domain is involved in protein-protein interactions with one  monomer, 1. The second  monomer, 2, does not interact with  and instead contacts '.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Structural context of the minimal  domain capable of  binding and  assembly mutations. The RIBBONS diagram of the three-dimensional structure of Taq RNAP core enzyme is shown (33). The  subunit is indicated in blue, ' in magenta, 1 in green, 2 in yellow, and  in white. The active center Mg2+ is shown in red and spacefill. The domain of  corresponding to E. coli resides 800-1231 is shown in cyan. A, view roughly parallel with the main axis of the RNAP channel. The active site Mg2+ is seen through the secondary channel. B, view A rotated 90° clockwise about the vertical axis. C, view A rotated 180° about the vertical axis, giving a view down the opposite end of the main channel. D, the RIBBONS diagram of Taq  fragment corresponding to E. coli -binding domain 800-1231(cyan) complexed with 1 (green). The catalytic Mg2+ is shown in spacefill and red. Parts of  segments F and I important for intrasubunit interaction are shown in magenta and orange. The 1 residue corresponding to E. coli Arg45 is shown in yellow and spacefill. E, the view in D was rotated approximately 90o counterclockwise about the horizontal axis. Taq  residues corresponding to E. coli residues corresponding to E. coli positions that when mutated alter  binding, as well the catalytic residues involved in substrate interactions are indicated, and shown in color and spacefill. Residues indicated in white correspond to E. coli  residues 1095, 1114, and 1228, and are not involved in  binding.

The -binding domain is held together by a 6-strand antiparallel -sheet. Conserved segments F, H, and I contribute to this  sheet, and stretches of amino acids corresponding to E. coli 800-812 and 1226-1231 each form interacting strands (Fig. 5D). We registered this intrasubunit interaction as a strong -galactosidase production when hybrids containing E. coli _800-907 and _911-1231 were combined. Removing either segment F or segment I amino acids disrupts the structure of the entire -binding domain, and as a consequence abolishes  binding.

Segment H and I Amino Acids Participate in  Binding-- The prototypical E. coli mutation that decreases the avidity of  binding to  changes  Arg⁴⁵ which is evolutionary conserved (12). We hypothesized that, in , the likely counterpart of  Arg⁴⁵ is negatively charged and evolutionarily conserved. Out of three possible candidates, all of which were located in segment H, only one, Asp¹⁰⁸⁴, when mutated affected  binding both in two-hybrid assay, and in in vitro co-immobilization experiments. In addition, substitution of segment I Gly¹²¹⁵ also abolished  binding.

The structure reveals that Taq Asp⁸⁵⁷, which is homologous to E. coli  Asp¹⁰⁸⁴, is indeed in direct contact with Arg⁴² (E. coli Arg⁴⁵) in 1 (Fig. 5E). Taq Gly⁹⁷⁷, which is homologous to E. coli  Gly¹²¹⁵, is located at the tip of a loop formed by segment I and buttresses Asp⁸⁵⁷ (E. coli Asp¹⁰⁸⁴) to position it such that it can correctly interact with Arg⁴² of 1. The G1215D substitution that we engineered probably causes unfavorable electrostatic interaction with Asp¹⁰⁸⁴ and may significantly alter the conformation of the -binding domain. In agreement with this idea, G1215D abolished the ' entry in the complex. In contrast, RNAP harboring  D1084A mutation assembled efficiently and was active. Thus, ' can stabilize the ₂ subassembly, presumably through independent protein-protein interactions with the second  monomer, 2, and . Of the remaining four mutations that we engineered, one (G1218D) substantially decreased  binding, while others had no effect. Taq Gly⁹⁸⁰ (E. coli Gly¹²¹⁸) is in the same loop as Taq Gly⁹⁷⁷ (E. coli  Gly¹²¹⁵); it makes no contacts with  and is at least 10 Å away from Arg⁴² (E. coli Arg⁴⁵). Thus, G1218D substitution inhibits -binding indirectly, probably by affecting the relative position of the stabilizing Gly¹²¹⁵. The importance of precise positioning of  Arg⁴⁵ and  Asp¹⁰⁸⁴ for ₂ formation is underscored by the fact that no interaction was detected when a pair of mutants containing an Asp in place of Arg⁴⁵ and an Arg instead of Asp¹⁰⁸⁴ was tested (data not shown).

The -binding domain of  also contains two strictly conserved residues that are implicated in catalysis, and that are exposed into the DNA-binding channel of the enzyme, close to the catalytic Mg ion. In segment H, Taq Lys⁸³⁸ (Lys¹⁰⁶⁵ in E. coli) contacts the -phosphate of the initiating purine nucleotide (6); in segment F Taq Glu⁶⁸⁵ (Gly⁸¹³ in E. coli) is probably required for proper interaction with incoming NTP (29). Binding of  thus stabilizes the active center conformation. In agreement with this idea, we had found that in the absence of ',  is necessary to induce specific binding of initiating purine nucleotide by the  subunit.²

Yeast RNAP Assembly-- Our studies of yeast RNAP subunit interactions reveal several important points. First, as expected, the determinants of  binding reside in the C-terminal portion of A135, the -like subunit of RNAP I, and Asp⁹³⁵, which is homologous to E. coli  Asp¹⁰⁸⁴ is required for this interaction. This result is in agreement with recent RNAP interaction mapping by Flores et al. (36). These authors reported that A135 segment 678-1055, containing conserved segments E, F, G, H, and I_C (corresponding to E. coli  positions 642-1253), bound both AC19 and AC40 in two-hybrid assay. Our interacting fragment of A135 contained conserved segments F, G, H, and entire I (amino acids 716-1201), and corresponded to E. coli  residues 681-1342. Based on mutational analysis, A135 appears to interact with the larger -like subunit, AC40, and not with the smaller AC19. This is consistent with structural analysis of yeast RNAP II by the Kornberg group (34), who observed that the largest -like subunit, Rpb3, contacts the -like Rpb2. Our data indicate that, in yeast, the interaction between the -like and the -like subunits of RNAP I is much weaker than the corresponding interaction in E. coli, and A135 mutations that abolish this interaction are viable. The corresponding interaction may be even weaker in case of RNAP III. We and others (36) were unable to detect any interaction between RNAP III -like subunit C128 and AC40/AC19 using two-hybrid assay. Additional RNAP subunits may strengthen the weak ₂-like complex in eukaryotes. Indeed, the AC40/AC19 heterodimer was shown to bind ABC10 (Rpb10) shared RNAP subunit (32). More recently, crystallographic analysis revealed that ABC10 and ABC10 (Rpb12) bind to the larger -like subunit Rpb3 in yeast RNAP II and anchor it on the -like Rpb2 (34). An alternative possibility would be that the eukaryotic ₂-like complex is stabilized by the entry of the largest, '-like subunit in the complex, similar to the situation observed with E. coli  D1084A mutation. This latter scenario envisions that the relative amount of RNAP I and III in the cell is chiefly determined by the joint availability of catalytic  and '-like subunits, which are specific, and not by the shared -like subunits.

	ACKNOWLEDGEMENT

We thank Lenore Neigeborn (Rutgers University, Piscataway, NJ) for two-hybrid plasmids and advice.

	FOOTNOTES

^* This work was supported in part by a Burroughs Wellcome Fund for Biomedical Research career award, by National Institutes of Health Grant RO1 59295, and by March of Dimes Birth Defects Foundation Research Grant FY99-479 (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^§ Supported by Waksman undergraduate fellowships.

^¶ A Henry Rutgers Scholar. Supported by a New Jersey Commission for Cancer Research summer fellowship.

^** To whom correspondence should be addressed: Waksman Inst., 190 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik@waksman.rutgers.edu.

Published, JBC Papers in Press, July 20, 2000, DOI 10.1074/jbc.M003884200

² T. Naryshkina, A. Mustaev, S. Darst, and K. Severinov, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; NTA, nitrilotriacetic acid; Rif, rifampicin; NTD, N-terminal domain; Gal4-DB, GAL4 DNA binding domain; Gal4-AD, GAL4 activation domain.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES