FEMSMicrobiologyLetters90 ( 199t) 57-62 © 1001Federationof European MicrobiologicalSocieties0378-1t)97/'9t/$1.~3,50 Publishedby Elsevier
RNA polymerase hetcrogeneity in Streptomyces aureofaciens" characterization by antibody-linked polymerase assay Marian Farkagovsk,~, Jfin Kormanec and Marta Koll,4rovfi Shn'ak Academy of Sciences, h~stit.te ~ffMolec'ularBiolot,%Bratisltoa, (':echt~slorakia Received3(ISeptember1091 Accepted 3 October 1901
Key words: Antibody-linked polymerase assay; RNA polymerase: Streptomyces aureofaciens
1. SUMMARY Using antibody-linked polymerase assay we studied the polypeptide composition of DNA-dcpendent RNA polymerase from Streptomyces attreofaciens and immunological cross-reaction with Escherichia colt RNA polymeras~:. We identified about 25 'ALPA-,eactive' polypeptides which are probably involved in the transcriptional apparatus. We demonstrated that B' and ,8 subunits from S. attreofaciens and E. colt are immunologically related and o"Tt~ (E. colt) shows immunochemical similarity with ~f.~5 (S. aureofaciens). According to the reconstitution of RNA polymerase holoenzyme and antibody-linked polymerase assay we identified sigma factors responsible for recognition of two promoters.
Correspondence to: M. Farka~ovsk)7.Institute of Molecular Biology,SlovakAcademyof Sciences,Dfiblavsk.'icesta21,842 51 Bratislava,Czechoslovakia.
2. INTRODUCTION The eubacterial DNA-dependent RNA polymerase holoenzymc (EC 22.214.171.124) containing five subunits (ot,/3~'o')is able to recognize promoter '~;equences and to polymerize ribonucleosidc triphosphates into RNA. Sigma subunit controls the specificity of the promoter-holocnzymc interaction and confers on the enzyme the ability to initiate transcription. The existence of multiple forms of RNA polymerase holoenzyme was presumed in an early work with this cnzyme . In the past fcw years this hypothesis was confirmed in different bacterial genera [2-5]. "Alternalive' holoenz3'me forms, carrying diffcrcnt sigma factors, recognize various classes of promoters required for the expression of the physiologically related genes. RNA polymerase heterogetteity was first proved in s~.reptomycetes by the work of Westpheling et a!. . They biochemically identified two Streptomyces coelicolor hoioenzymes, each recognizing in vitro Bacillus subtilis promoters of
a different class. Three different holoenzymcs of RNA polymerase from S. coeficolor were separated by Buttner el al. , using fast protein liquid chrematography, each of which transcribed only from one of the four tandem promoters of the dagA gene (encodes extracellular agarase). This discovery added cr-'~ to the two sigma factors, o"~~ and o-4', identified by . Tanaka et al.  used another approach by which four genes for the principal sigma factor were idenlified in S. coelicolor. They used a synthetic oligonucleotide probe corresponding to a conserved sequence in the rpoD gene product of L~cherichia coil. Streptomycetes are unusual among prokaffores in the complexity of their morphological differentiation cycle. The process of sporulation of aerial hyphae requires at least eight sporulation-specific genetic loci (whi genes) . The whig appears to i:,c-a--k,~y-gonein the triggering of the onset of sporulation in aerial hyphae. The amino acid sequence of the whiG product shows a striking similarity to the sequences of sigma factors . In the present paper we applied another approach to analyze heterogeneity of RNA polymerase in Streptomyces species. Using an antibody-linked polymerase assay [I 1,12] we studied DNA-dependent RNA polymerase of Streptomyc~s aureofaciens with regard to the polypeptide composition and immunological cross-reactio.n with E. coli RNA polymerase.
3. MATERIALS AND METHODS
3.1. Bacterial strabz, media and culture cond#ions For the isolation of RNA polymerase Streptomyces aureofaciens 2201  was grown to early stationary phase in GAFSK medium  at 30°C in 5-1 shake flasks.
3.2. Preparation of RNA polymeras¢ antisera and antibody-linked polymerase assay (ALPA) RNA polymerase was isolated from S. aureofaciens 2201 by Polymin P precipitation, salt extraction, ammonium sulfate precipitation, DNAagarose affinity chromatography and Superose 6 gel filtration as previously described [7,14]. The
E. coli RNA polymerase was purchased from Boehringer (Mannheim, F.R.G.). Rabbit antiserum against S. aureofaciens RNA polymerase (from the last step of isolation) was prepared as described in . Protein blotting and ALPA were performed according to [11,12], with the exception that between the binding reactions of ALPA more extensive washing procedures were used. Transfer of polypeptides was proved by colloidal gold staining .
4. RESULTS AND DISCUSSION
4. I. Characterization of RNA polymerase from £ aureofaciazs by ALPA ALPA was introduced by Van der Meer et al.  with the aim of correlating RNA-dependent RNA polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel without renaturation of protein and was based on a solid-phase 'Sandwich' enzyme immunoassay on nitrocellulose filter strips . After blocking of the free-binding sites of the nitrocellulose filter with bovine serum albumin antibodies against RNA polymerase (and other polypeptides) are linked to nitrocellulose-bound polypeptides via Fab-site (first binding reaction). In the second binding reaction native polymerase molecules are bound via their corresponding subunits to the other Fab-sites of the antibody moIecules (also non-polymerase polypetides, which are present in partially purified polynierase, are bound). After transcription with immobilized enzyme the radioactively labelled transcription products are precipitated in situ with trichloroacetic acid. The merit of this method is that the use of both non-specific antiserum and a partially purified enzyme is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel eI,~ctrophoresis. Application of ALPA to a multimerle enzyme presumes that every enzyme subunit in the native enzyme is available to antibodies and that native enzyme bound to immobilized antibodies in the second binding reaction does not lose the enzyme activity. These two conditions should be fulfilled by using native enzyme for antibody preparation .
For ~:',trocellulose blots and binding of native enzyme in our initial experiments we used RblA polymerase after two steps of purification: PE! fractionation (crude) and Superose 6 gel fil',ration (purified). However, in furlher studies we preferred crude RNA polymerase for the following reasons: (a) the possibility of partial proteolytic degradation of the enzyme increases during purification and, (b) potential loss of transcription factors during the purification procedure. For the preparation of antiserum we used RNA polymerase from the last step of isolation to decrease the level of non-RNA polymerase antibodies which are also generated and can complicate the analysis. On the other hand purified RNA polymerase used as immunogen contains fewer minor transcription factors than the crude one. Experiments using anti-rabbit lgG-horseradish peroxidase conjugate (Amersham) to detect immune complexes showed that our poly-specific rabbit antiserum contains antibodies against about 45 polypeptides (Fig. 1A). This result allows us to suppose that our antiserttm also contains antibodies against minor transcription factor. Transcription factors are associated with the core enzyme for a limited period, tirerefore we used only 3 pmol [a-3ZP]CTP (Amersham, 3000 Ci/mmol) and a short period of transcription (I-4 rain) to arrest transcription before the transcription factor is released from the enzyme. Under these conditions about 25 'ALPA-reactive' polypeptides were identified. The possibility of using the ALPA to determine transcription factors like o" was proved previously by using E. coli RNA polymerase as a model system . Several 'ALPAreactive' polypeptides have the same molecular masses as the previously biochemicatly identified subunits and factors of streptomycetes RNA polymerase: ,8, /3', a, o-~5, o"4~, o~'8 [6,7,18]. Polypeptides with molecular masses of 35000 (o-3~), 52000 (o-+~) and 28000 (o":~) are also copurified with the o~,/3 and/~' subunits and with polymerase activity. This supports the idea that these polypeptides are sigma factors (data not shown). The molecular mass of these polypeptides was determined by comparing their dectrophoretic mobility with that of the protein standards (Pharmacia low- and high-molecular mass
67 - ¸
Fig. !. Determination of 'AEPA-reactive' polypeptides of crude S, aureofaciens RNA potymerase. Nitrocellulose blots of crude RNA polymerase were treated as follows: lane A: incubation with antiserum and anti-rabbit IgG+horseradish peroxida~e conjug~lte Ienzyme reaction with DAB substrate): lane B: incubation with antiserum and crude S. aureofaciens RNA polymerase followed by the transcription reaction: lanes C, D, E, F: control experiments as iadicated in the text; lane G: incubation with antiserum and E. coil RNA polymerase fork~wed b3, the tr,'mscription reaction. Relative kDa marked at left.
calibration kit) cn nitrocellulose blot stained with colloidal gold or on silver-stained polyacrylamide gels (not shown). Several control experiments were done with the aim of recognizing possible artefacts: (a) transcription reaction was performed without template DNA (Fig. IC). a, /3' and '8 subunits gave weak signals which is probably due to the presence of traces of DNA in the crude RNA polymerase preparation; (b) direct
poiymerasc assay without incubation with antiserum and native enzyme excluded binding of the substrate [a-“P]CTP to the poiypeptides on the nitroceiiuiose filter (Fig. 1F); (c) binding of native RNA poiymerase to the poiypeptides on the nitroceiiuiose blot via DNA was ruled out by subsequent incubation with antiserum and nick-translated S. aureofaciens 2201 chromosomal DNA (ten times more than in transcription mix) (Fig. 1D); (d) to exclude binding of native RNA poiymerase to polypeptides on the nitroceiiuiose blot without the mediation of the specific antibody, the nitroceiluiose filter was incubated with preimmune serum and successively incubated with native enzyme (Fig. 1E). Washing procedures in contrai experiments were performed after each binding reaction as in ALPA. Artefacts were not visible even after prolonged exposure of the autoradiograms. According to the results described above, we suppose that ‘ALPA-reactive’poiypeptides, if not proteolytic products of RNA poiymerase subunits (factors), are involved in transcriptionai apparatus. 4.2. Immurrological cross-reaction of E. coii RNA polymerase and S. aureofaciens RNA polymeruse Both E. coli and S+aureofuciens RNA poiymerase belong to the class of eubacterial RNA poiymerases with similar subunit composition ((u, /3’, /3, ~1. These subunits contain highly conserved regions, which can also cause their immunochemicai similarity. In experiments where nitroceiiuiose blots of E, cull RNA poiymerase were subsequently incubated with antiserum against streptomycete RNA poiymerase and streptomycete native enzyme, three ‘ALPA-reactive’ E. coii polypeptides are observed: /3’, /? and a” (Fig. 2C). The (Ysubunit of E. coli poiymerase was not ‘ALPA reactive’ even after prolonged exposure of the autoradiograrrls or after changes in conditions of the transcripticlnor binding reactions. However, when nitrocelll.doseblots of streptomycete RNA polymerase were incubated first with antiserum against strsptomycete RNA poiymerase, then with native RNA poiymerase from E. co/i, seven ‘ALPA-reactive’ poiypeptides were identified: /3’, /3, a, g3’ and polypeptides of 45 kDa, 41 kDa and 40.5 kDa
Fig. 2. Immunological cross-reaction of E. co/i and S. aureofaciemRNA polymerase. Lane A: silver-stained polyacrylamide gel: lane B: nitrocellulose blot of E. cult’ RNA polymerase stained with colloidal gold: lane C: incubation of nitrocellulose blot of E. coli RNA polymerase with antiserum and 5, aureofhwsRNA polymerase followed by the transcription reaction; lane D: control experiment (as lane C except that the transcription reaction was performed without template DNA); M: marker proteins (Pharmacia). Relative kDa marked at left.
(weak signals, Fig. 1G). These experiments showed that /3’ and j3 subllnits from E. co/i and S. aureofuciens are immunoIogicaiiy related. This is not a startling results, because /3’ and p subunits of eubacterial RNA poiymerases and similar subunits of eukaryotic and archaebacteriai RNA polymerases form homologous protein families [19,20]. Many eubacteriai ff factors show suggcstivc simi!arities in amino acid sequences .In immunological cross-reactions g7’ from E. co/i and r3’ from S. aureofacienswere detected (Figs, 2C and ZG) and this confirms the idea that g3’ is a major o‘ factor in streptomycetes [6,7]. Absence of the signal of E. coli LYsubunit (Fig. 2C) probably indicates low immunological relation between E. coli and S. aureofaciens LYsubunits. 4,3, Identificationof sigma subunits recognizing promoters of different classes and further perspect&es of ALPA Cautious interpretation of the results of ALPA is necessary, because one cannot exclude subsequent binding of the antibody and native RNA
61 polymerase to the polypeptide which is immunologically related to the particular subunit or factor but is not involved in transcription. However, based on control experiments (Fig. 1, lanes C, D, E, F) we can presume that most of the 'ALPA-reactive' polypeptides are involved in the transcriptional machinery and are potential candidates for new transcription factors. By the analysis of the previously described promoter region , which is probably involved in expression of the genes responsible for the production of an antibiotic compound, we used in vitro transcription with reconstituted holoenzyme (RNA polymerase) to identify sigma factors responsible for recognition of promoters in this region. Crude RNA polymerase was subjected to SDS-polyacrylamide gel electrophoresis and the region of the gel, containing proteins in the relative molecular mass range 25000-100000, was horizontally cut into eight slices. Proteins from these gel pieces were eluted and renatured according to the method of Hager and Burgess . Reconstitution of the holoenzyme and in vitro transcription was performed as described in . The 222-nucleotide (nt) band reflects end-to-end transcription of the DNA template (Fig. 3, lane 1-8). 129 nt band (Fig. 3, lane 6) corresponds to expected transcript from promoter A1  and the 164-nt band (Fig. 3, lane 5) corresponds to a transcript from promoter (A2) which is located 26-n,t!upstream of A1 promoter (gives a weak signal by S1 nudease mapping when compared with A1 promoter). The only detectable polypeptide by the method ALPA in gel slice 5 was of relative molecular mass 52000 previously described as tr 49 [6,7]. Thus this sigma factor is responsible for recognition of this promoter. In the case of A1 promoter, we repeated reconstitution of holoenzyme with renatured polypeptides from the region of gel slice 6 horizontally divided in several parts, because there are more than one 'ALPA-reactive' polypeptides in this region. Result of this experiment enables us to determine that polypeptide with relative molecular mass 35 000 (tr 35) is responsible for recognition of this promoter (data not shown). ALPA gives an idea about complexity of the transcriptional apparatus and can serve as a suit-
Fig. 3. Reconstitutionof RNA polymeraseholoenzyme.Runoff transcripts were generated from 222-bp long EcoRlHindllI fragment of recombinant plasmid pFK 13  with reconstituted RNA polymerase holoenzymes.Products were analyzed on 6% polyacrylamide-7M urea gels. The lane numbers correspond to the gel slicesfromwhich the polypeptides were eluted and renatured. LaneM, [32P]-labeledHaelll digestof ~X-t74.
able method for studying changes in the composition of RNA polymerase from bacteria grown under diffent physiological conditions. ALPA is also a valuable assay method for the isolation of potential candidates of transcription factors (for N-terminal or internal sequence analysis). Such studies are in progress.
ACKNOWLEDGEMENTS We thank E. Brfiutigam for her valuable advice. Critical reading of the manuscript by L. Potu(:kova is gratefully acknowledged.
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