Structure of p factor : An RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains ( transcription termination factor / chemical modification / domain structure / protein cleavage / ATPase )

Content Provider	Semantic Scholar
Author	Dombroski, Alicia J. Platt, Terry
Abstract	The domain structure of p protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for p function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (Ni), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDodS04/polyacrylamide gels and renatured. The Ni fragment binds poly(C) and mRNA corresponding to the p-dependent terminator sequence tip t', but not RNA unrecognized by p; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved p renatures to regain its RNA-dependent ATPase activity, but neither Ni nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the a subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotidebinding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination. p protein of Escherichia coli is a transcription termination factor that catalyzes 3' endpoint formation and release of mRNA molecules from DNA templates (1-3). This essential protein has identical subunits of 419 amino acids (4, 5) and exists as a hexamer in solution (5, 6). p binds to both single-stranded RNA and single-stranded DNA (7, 8); its highest known affinity is for synthetic polycytidylate [poly(C)] (9). When activated by binding to RNA, p has the ability to hydrolyze ATP in the absence of other transcriptional components (9, 10); this RNA-dependent ATPase activity is also necessary for transcription termination (11). We recently demonstrated that the RNA-dependent ATPase activity is crucial as well for the RNA-DNA helicase activity of p (12). This activity most likely serves the observed transcriptional function of releasing completed mRNA from the DNA template (13, 14). Our understanding of the functional mechanisms by which p catalyzes transcription termination is thus becoming clearer, yet our knowledge of the structural determinants necessary for RNA binding and ATP hydrolysis, and the interactions between them that are required for termination, is still very limited. p's RNAdependent ATPase activity (essential for termination) can be inactivated by reagents that react with free sulfhydryl groups (15), and one attractive possibility for RNA recognition proposed that a transient covalent intermediate (termed a Michael adduct) might form between the sole cysteine at position 202 in each p subunit (4) and regularly spaced cytosine bases in the RNA substrate to "nucleate" p binding (1). In this paper we demonstrate that Cys-202 is completely dispensable for p function, thereby unequivocally ruling out the Michael adduct hypothesis for recognition. We report the results of attempts to define more precisely the RNAbinding and ATPase regions within the p protein and discuss how the RNA-binding domain may interact with an adjacent ATP-binding domain to catalyze RNA-dependent ATP hydrolysis and transcription termination. MATERIALS AND METHODS Oligonucleotide Site-Directed Mutagenesis. The p gene was removed from plasmid p39-AS (16) by cleavage with restriction endonucleases Bcl I and HindIII and subcloned into the BamHI and HindIII sites of pUC118 (a phage M13-derived vector from J. Vieira, Waksman Institute, Rutgers, NJ). The resulting plasmid, pAJD1, was used to transform competent E. coli strain RZ1032 ung, dut (17). Single-stranded plasmid DNA containing uracil was generated by infection with the helper phage R408, from Stratagene (San Diego, CA). Synthetic oligonucleotides d(CCGGATAGTGTGCTGATG) (for the cysteine-to-serine mutation) and d(CCGGATGGTGTGCTGATG) (for the cysteine-to-glycine mutation) were prepared with a DNA synthesizer (Applied Biosystems, Foster City, CA). The two-primer method (18) was used for mutagenesis in vitro, and the resulting double-stranded DNA molecule was transformed into E. coli strain BMH71-18 mutL (19), which is defective for mismatch repair, to improve the efficiency of mutagenesis. Colonies were probed by colony hybridization (20) with the same oligonucleotide used for mutagenesis but now 32P-labeled at the 5' end. Single-stranded DNA from positive colonies was used as template for 35S/dideoxy sequencing (21, 22). Overproduction of Proteins. The mutated p genes were removed from pAJD1 on an EcoRV restriction fragment of -1800 base pairs and ligated into the EcoRV restriction sites of p39-AS. These plasmids were transformed into E. coli strain AR120-A6, a dam::Tn9 derivative of AR120 (16). Nalidixic acid induction (16) yielded high levels of mutant p proteins that were partially purified by centrifugation on sucrose density gradients. Approximately 1 g of cells was broken in a French pressure cell press and stored frozen; extracts were thawed, resuspended to 2 ml in buffer A (23), and microcentrifuged for 15 min at 40C. The supernatant was diluted to 1.5 ml with the same buffer and 0.1 ml was loaded onto each 5-ml sucrose gradient (5-20% sucrose). The gradients were run in a Beckman SW50.1 rotor to w2t = 6 x Abbreviations: NTCB, 2-nitro-5-thiocyanobenzoic acid; pHMB, p-hydroxymercuribenzoate; MaINEt, N-ethylmaleimide. 2538 The 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. Proc. Natl. Acad. Sci. USA 85 (1988) 2539 1011 at 40C. Nine fractions were collected from the bottom of the gradient and analyzed by 0.1% NaDodSO4/10% polyacrylamide gel electrophoresis, and the peak p fraction (second from bottom) was chosen for subsequent analyses. Protein Analysis in Vitro. For RNA-binding studies poly(C) was either 5'or 3'-end-labeled, purified by electrophoresis in 7 M urea/10% polyacrylamide gel, eluted by soaking in 0.3 M sodium acetate/0.1 mM EDTA, filtered, precipitated, and resuspended in 50-75 A.l of water. Binding of p protein to 32P-labeled poly(C) was carried out in ATPase assay buffer plus 0.5 mM ATP, 0.9 nCi (33.3 Bq) of 32P-labeled poly(C) [100 nM in nucleotides, or 1 nM poly(C) with a range of lengths averaging about 100 nucleotides], and 0-75 nM p protein in a total reaction volume of 50 Al. The mixture was incubated at 37°C for 20 min, followed by filtration of 45 ,ul through 13-mm-diameter nitrocellulose filters (Schleicher & Schuell). The filters were washed twice with 45 ,l of ATPase buffer, dried briefly, and subjected to scintillation counting to measure the RNA bound to p. Synthesis of the p-dependent termination sequence trp t', and of its complementary mRNA in vitro by use of SP6 RNA polymerase, has been described (12). Chemical Modification of p Protein. p protein was cleaved at Cys-202 with 2-nitro-5-thiocyanobenzoic acid (NTCB; Fig. 1) (24), by adding NaDodSO4 (final concentration 0.1%) to 100 ,l of p from gradient fraction 2 plus 10 Al of 37 mM NTCB (in ethanol) and incubating at 37°C for 30 min. Then 100Al of 1 M acetic acid was added, followed by 1 ml of ice-cold acetone. The protein was precipitated by microcentrifugation for 5 min at 4°C. The pellet was washed twice with 0.5 ml of cold acetone, dried, and resuspended in 20 ,l of 1% NaDodSO4. One hundred microliters of 0.1 M sodium borate (pH 9.6) was added and the reaction mixture was incubated for 14-16 hr. Products were analyzed by 0.1% NaDodSO4/10% polyacrylamide gel electrophoresis. Uncleaved p polypeptide chains and the cleavage fragments were eluted from polyacrylamide gels and renatured according to Hager and Burgess (25). Cleavage at Asn-151 by hydroxylamine was performed in a reaction mixture containing 70 nM p, 1.7 M NH2OH (dissolved in 2 M Tris.HCI/2 M LiOH), and 0.2% NaDodSO4. Following incubation at 45°C for 3 hr, acetic acid was added to 0.5 M. The protein was precipitated using ice-cold acetone (as for the NTCB cleavage), dried, and resuspended in 50 ,l of 0.8% NaDodSO4. Fragments were eluted and renatured in the same manner as for NTCB cleavage. Modification of p with N-ethylmaleimide (MalNEt) was achieved in reaction mixtures consisting of 70 nM p from gradient fraction 2, ATPase buffer, and 100 mM MalNEt. Cys-202 in p was modified by adding phydroxymercuribenzoate (pHMB, dissolved in 0.1 M NaOH
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