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ROBERT LANDICK§, VADIM NIKIFOROVt, ALEX GOLDFARBt, AND SETH A. DARST*¶ ...... Burbaum, J. J. & Schimmel, P. (1991) Biochemistry 30, 319-324. 49.
Proc. Natl. Acad. Sci. USA

Vol. 92,

pp. 4591-4595, May 1995

Biochemistry

Assembly of functional Escherichia coli RNA polymerase containing (8 subunit fragments KONSTANTIN SEVERINOV*, ARKADY MUSTAEVt, ELENA SEVERINOVA*t, IRINA BASSt, MIKHAIL KASHLEVt, ROBERT LANDICK§, VADIM NIKIFOROVt, ALEX GOLDFARBt, AND SETH A. DARST*¶ *The Rockefeller University, New York, NY 10021; tThe Public Health Research Institute, New York, NY 10016; tInstitute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia; and §Washington University, St. Louis, MO 63130 Communicated by Carol A. Gross, University of California, San Francisco, CA, February 9, 1995 (received for review November 15, 1994) show that the 3 subunit split at dispensable region I and at the two dispensable regions simultaneously can still assemble into a functional enzyme in vitro. These results indicate that the 3 subunit is composed of at least three independent domains and delineate a strategy that can be used to define further the domain architecture of the RNAP subunits.

The Escherichia coli rpoB gene, which codes ABSTRACT for the 1342-residue j3 subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP 1 subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these p-subunit polypeptide fragments, when added with purified recombinant P', or, and a subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the 1f subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.

MATERIALS AND METHODS

Sequence comparisons among eubacterial, archaebacterial, eukaryotic P-subunit homologues reveal regions of high sequence similarity separated by regions that are poorly conserved (6, 21-23). Indeed, in some organisms the nonconserved regions contain large gaps or insertions compared with the E. coli ,3 subunit. Deletions in two of these regions, centered around residues 300 and 1000 and termed dispensable regions I and II, respectively, do not affect RNAP assembly and basic function in vitro (24, 25). The two dispensable regions contain >25% of the total (3-subunit length. In addition, physical continuity of the ( subunit in dispensable region II is not necessary for RNAP assembly and basic function in vitro (26). In this report we extend these studies and

Techniques. The pUC19-based pMKA92 rpoB expression plasmid has been described (27). The rpoB frameshift mutations originating from Alu I and Taq I restriction sites were constructed in the pMKA92 plasmid as follows. The plasmid DNA was linearized by random cuts with either Alu I or Taq I restriction endonucleases, and termini were filled-in by using the Klenow fragment of DNA polymerase I. The DNA was ligated with the EcoRI fragment (with termini filled-in with the Klenow fragment of DNA polymerase) from phage mpl7Km carrying the kanamycin-resistance gene. This procedure was expected to destroy the rpoB reading frame and to generate t polypeptide amber fragments terminated shortly after the kanamycin cassette insertion site. Kanamycinresistant transformants were screened for the appearance of inducible truncated fragments of the P polypeptide by SDS/ PAGE. The size of these fragments corresponded to the known positions ofAlu I or Taq I restriction sites within the rpoB gene and to the location of kanamycin inserts determined in each case by restriction mapping. The plasmid expressing 3-(235-1342) was obtained from the in-frame BamHI linker insertion at position 235 of rpoB cloned in the pMKA92 plasmid (25). The plasmid was treated with BamHI and Sma I (which cuts once in the polylinker after the structural gene) and the fragment containing most of rpoB was recloned into pUC19. Recombinants were selected for the appearance of inducible truncated fragments of the ( polypeptide by SDS/PAGE. The resulting plasmid was used to construct a (3-(235-989) expression plasmid by replacing the of rpoB with a fragment from a plasmid corresponding part expressing the 3 amber fragment P-(1-989). E. coli XL1-blue was used as the expression host for all the plasmids described above. The plasmid expressing 8-(950-1342) was obtained by PCR amplification of the relevant portion of the pMKA92 rpoB gene and cloning the amplified product into the pET15b expression vector (Novagen). The fragment was then exin BL21 (DE3) E. coli cells. All of the overexpressed pressed ( fragments were found in inclusion bodies when induced with 1 mM isopropyl 3-D-thiogalactoside at 37°C. RNAP Reconstitution. RNAP was reconstituted as described (28), with modifications to be described elsewhere (54). The molar ratio of a and 3' in the reconstitution reactions was 1:4, and each (3-subunit fragment was added to 8-fold

publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: RNAP, RNA polymerase. 1To whom reprint requests should be addressed.

Genetic

DNA-dependent RNA polymerase (RNAP) is the central enzyme of gene expression and a major target for genetic regulation. The structure of cellular RNAP has been highly conserved during evolution (1-3), as documented by the high level of "similarity between the primary sequence of RNAP subunits from bacteria to humans (4-8). Recent models of RNAP function postulate a substantial degree of intramolecular motion within the transcription complex during transcription (9-11). Movement of enzyme parts relative to each other could be realized by structurally independent and functionally important domains that are connected by flexible loops or hinges. Identification of such domains would afford insight into the mechanism of RNAP function. Most information about RNAP structure and function comes from studies of Escherichia coli RNAP. The ( subunit (150.6 kDa) is highly conserved throughout evolution (6, 8, 12). Like the other RNAP subunits, the 3 subunit in isolation has no apparent function. When assembled in the RNAP complex, 3-subunit residues 516-540, 1065, and 1237 participate in the formation of the initiating site of the enzyme (ref. 13; K.S., A.M., E.S., M. Kozlov, S.A.D., and A.G., unpublished data). Mutations in the 3 subunit render the enzyme resistant to the inhibitors rifampicin (residues 144-146, 511-534, 567574, and 687; refs. 14-18) and streptolydigin (residues 540546; refs. 15, 19, and 20).

and

molar excess relative to the a subunit. After reconstitution and

The

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Proc. Natl. Acad Sci. USA 92 (1995)

thermoactivation in the presence of a70 purified from a superproducing strain (29), RNAP preparations were either directly used for affinity labeling or further purified by FPLC gel filtration on a Superose 6 column (Pharmacia LKB) as described (28), concentrated by filtration through a C-100 concentrator (Amicon) to -1 mg/ml, and stored in 50% glycerol storage buffer at -20°C. Affinity Reagents, Affinity Labeling, and Cross-Linked Product Analysis. Synthesis of the aldehyde derivative of AMP is as described (30). For affinity labeling, 20 ,1I of reconstitution mixture was supplemented with 1 t,g of purified o070. After incubating for 15 min at 37°C, initiating substrate analog was added to 0.5-1.0 mM. Reactions were supplemented with 100 ng of a 136-bp T7-A1-promoter-containing DNA fragment (31), obtained by PCR amplification, and incubated for an additional 15 min at 37°C. Sodium borohydride was added to 10 mM and incubation at 37°C was continued for 10 min. Reaction mixtures were transferred to room temperature and incubated for 30 min with 0.3 ,tM [a-32P]UTP (3000 Ci/mmol; 1 Ci = 37 GBq). Reactions were terminated by adding an equal volume of SDS-containing Laemmli loading buffer and proteins were resolved by SDS/PAGE in precast Tris-glycine gels (NOVEX, San Diego). Gels were stained with Coomassie blue and affinity-labeling products were visualized by autoradiography. Control experiments demonstrated that 32P-labeling of RNAP subunits depended on the addition of template DNA. In Vitro Transcription. The 444-bp DNA fragment with Pr7Altrp-trmBT1 used for transcription experiments was prepared by PCR of a derivative of the pRL407 plasmid (32). Transcription complexes containing radiolabeled 21-mer transcript were prepared in solid-state transcription reactions by incubating 1 pmol of immobilized RNAP (33) with 10 pmol of template DNA, 0.5 mM ApU, 50 ,tM ATP, 50 ,tM CTP, and 50 ,tM GTP, and rifampicin (5 ,tg/ml) for 5 min at 37°C. Reaction products were washed to remove unincorporated substrates (33) and incubated for an additional 5 min with 0.5 ptM [a-32P]UTP (3000 Ci/mmol) at room temperature. After washing, imidazole was added to 100 mM and transcription complexes were allowed to desorb for 15 min at 4°C, with occasional mixing. After a brief low-speed centrifugation, aliquots of supernatant were used for chain extension with all four NTPs (each at 100 ,uM) in the presence or absence of recombinant Alc protein (34).

RESULTS Generation of P-Subunit Fragments and Strategy for in Vitro Functional Analysis. A collection of random kanamycin cassette insertions in the rifampicin-resistant rpoB gene allele N516Y cloned on the pMKA92 expression plasmid (27) was used to obtain the 3-subunit fragments used in this work. The overexpressed 13 fragments were recovered from inclusion

bodies by using guanidine hydrochloride-containing buffer, intact a and 13' subunits were added, and the denaturant was dialyzed away under conditions favoring RNAP assembly from intact a, 3, and 3' subunits (28). The function of reconstituted RNAPs was tested by using promoter-dependent affinity labeling of the 13 subunit (30). In this assay, RNAP is derivatized with an initiating (+ 1) nucleotide analogue, which is then extended in a templatedependent manner with radioactively labeled nucleoside triphosphate specified by position +2 of the template. As a result, a radioactive dinucleotide is covalently attached to the 13 subunit, which can then be visualized by autoradiography after denaturing electrophoretic separation. Labeling of the subunit requires catalytic activity of the RNAP since the enzyme must bind promoter DNA and catalyze formation of the first phosphodiester bond. A derivatized adenine nucleotide specific for RNAP 13-subunit Lys1065 was used (13). Therefore, radioactive labeling of 13 fragment(s) containing this residue is a sensitive, specific, and stringent test of the functional capacity of RNAP reconstituted with 13-subunit fragments. This functional test is not compromised by contamination of the preparations with wild-type RNAP, which is revealed, if present, by labeled full-size 3 on the gel. p Subunits Split at Dispensable Region I Assemble in Vitro into Functional Enzymes. Results of highly selective affinity labeling in reconstitution mixtures containing 3 subunits split at dispensable region I are presented in Fig. 1A. In this experiment, C-terminal fragment 13-(235-1342) was tested against a panel of N-terminal 13 fragments with C termini ranging from residues 186 through 433. Intact 3 was used as a positive control (Fig. 1A, lane 8). Radioactive labeling of 13-(235-1342) verifies its assembly into functional enzyme. ,3-(235-1342), in the absence of any other 3 fragments or in combination with the shortest N-terminal fragment, 13-(1186), did not result in detectable modification (Fig. 1A, lanes 6 and 7). All the longer N-terminal fragments tested supported modification of P-(235-1342). 3-(235-1342) labeling was still

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A

I

Split site:

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site: Spt 0

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J 9' PX. P235-1342-

I235-1342y-

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FIG. 1. Splitting the 3 subunit in its dispensable regions. Recombinant 3 fragments were incubated with 3', a, and subunits under conditions favoring RNAP reconstitution. The resulting preparation was then modified with the aldehyde AMP derivative specific for Lys1065 of the , subunit. The cross-link was then "developed" in the presence of [a-32P]UTP and reaction products were separated by SDS/PAGE. (Left) Gels stained with Coomassie blue. (Right) Cross-linked polypeptides visualized by autoradiography. (A) Splitting (3 in dispensable region I (upper gels, 6% polyacrylamide; lower gels, 12% polyacrylamide). C-terminal fragment 3-(235-1342) and the indicated N-terminal fragments (denoted by their C termini) were mixed in reconstitution reactions. In addition to the labeled bands [f3', P, 3-(235-1342), a, a, and X, which is a contaminant with a mobility slightly higher than a present in all the reconstituted RNAPs], the 3-subunit N-terminal fragments are visible in the 6% gel. 3-(1-308) fortuitously comigrates with a and f3-(1-276) has a mobility very close to band X. (B) Splitting 3 in dispensable region II and at both regions simultaneously. Wild-type 3 subunit (lanes 1), 3-(1-235) + 3-(235-1342) (lanes 2), f3-(1-989) + 3-(950-1342) (lanes 3),and 3-(1-235) + 3-(235-989) + 8-(950-1342) (lanes 4) were mixed in reconstitution reactions. Tris-glycine SDS/PAGE in 8% gels was used to resolve the reaction products. a

Proc. NatL Acad. Sci. USA 92

Biochemistry: Severinov et al.

shown).

fp Subunits Split at Dispensable Region II Assemble in Vitro into Functional Enzymes. Results of affinity labeling in reconstitution mixtures containing (3 subunits split at dispensable region II are presented in Fig. 1B. In this case, RNAP function is manifested by radioactive labeling of the shorter C-terminal fragment (3-(950-1342), which contains Lys1065, the residue modified in the affinity-labeling protocol. It was shown (26) that RNAP assembles and functions in vitro by using (3-subunit fragments produced by proteolytic cleavage, at around residue 1000, of genetically modified (3. The results shown in Fig. 1B, lanes 3, confirm this result by showing that functional RNAP was reconstituted from individually overexpressed recombi-

P-(950-1342).

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Transcription Elongation by RNAPs with Split P3. The affinity-labeling assay tests the ability of the reconstituted RNAPs to form only the first phosphodiester bond between the initiating nucleotide analog crosslinked to Lys1065 and the radioactive nucleotide specified by the +2 position of the template DNA. To test for transcript elongation, the reconstituted RNAPs were assayed for transcription of a DNA fragment containing the T7 Al promoter in vitro (Fig. 3). To exclude the possibility of contamination with wild-type en-

detectable when reconstituted with the large N-terminal frag13-(1-989) (750-residue overlap) with the extent of modification comparable to that seen in Fig. 1B, lanes 1 (data not

ment

nant N-terminal fragment

(1995)

zyme, RNAP split in dispensable region II or in both dispensable regions was first immobilized on Ni2+ nitrilotriacetic acid-agarose through a tag of 6 histidines positioned at the N terminus of 3-(950-1342). After extensive washing with transcription buffer, template DNA was added and an initial ternary complex containing a 21-nt transcript was generated. The amount3 of the 21-mer complex formed by RNAP with double-split subunit (lane 10) was similar to wild-type RNAP (lane 1). Purified starting complexes were allowed to resume elongation and transcribe through the strong trpL pause, the trpL attenuator, and finally the p-independent rrnB terminator (32). Most importantly, all three RNAPs reconstituted from P3-subunit fragments (split site in dispensable region I, split site in dispensable region II, or double-split 3B) were able to elongate the RNA transcript past the initially formed 21-mer. Fig. 3, lanes 2-9, shows a band of comparable intensity at the trpL pause site, indicating that all the enzymes elongated the RNA 81 nt to this position. Elongation past this site by RNAP reconstituted from P3-subunit fragments split at dispensable region II was essentially identical to control wild-type enzyme (Fig. 3, lanes 2 and 6). RNAP reconstituted from (3-subunit fragments split at dispensable region I did not elongate RNA past the trpL pause as efficiently as wild-type enzyme. Com-

(f-(1-989) and C-terminal fragment

p1 Subunits Split Simultaneously at Both Dispensable Regions Assemble in Vitro into Functional Enzymes. Results of affinity labeling in reconstitution mixtures containing 3 subunits split into three fragments, with the two split sites in dispensable regions I and II, are also presented in Fig. 1B. Reconstitution using the three fragments P-(1-235), P-(235989), and 3-(950-1342), in the presence of the other RNAP subunits, resulted in assembly of functional enzyme, as indicated by labeling of 3-(950-1342) (Fig. 1B). The presence of both (3-(1-235) and (3-(235-989) was required for affinity labeling of 3-(950-1342) (data not shown). The results of affinity labeling for all the reconstitution mixtures are summarized iii Fig. 2.

Breakpoint in archaebacteria Dispensable Region II Lvsios5

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100

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site

900