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Positive Factors Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8958-8962, October 1991 Biochemistry Functional map of the a subunit of Escherichia coli RNA polymerase: Two modes of transcription activation by positive factors (positive control/subunit assembly/transcription factor/protein-protein contact/deletion mutant) KAZUHIKO IGARASHIt, AKEMI HANAMURAt§, Kozo MAKINO¶, HIROFUMI AIBAII, HIROJI AIBA*§, TAKESHI MIZUNOII, ATSUO NAKATA¶, AND AKIRA ISHIHAMAt** tNational Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411, Japan; *Tsukuba University, Department of Chemistry, Tsukuba, Ibaraki 305, Japan; 1Osaka University, Institute of Microbial Diseases, Suita, Osaka 565, Japan; and 1Nagoya University, School of Agriculture, Chikusa, Nagoya 464, Japan Communicated by Motoo Kimura, June 24, 1991 ABSTRACT The role of the a subunit of Escherichia coli subunit is involved in positive control by certain transcription RNA polymerase in transcription activation by positive factors activators (10-14). was investigated using two reconstituted mutant RNA poly- In the present study, we extended this line of experiments merases (containing C-terminally truncated a subunits) and to type II CRP-dependent promoters. Most CRP-dependent three positive factors [the cAMP receptor protein (CRP), promoters can be classified into two types depending on the OmpR, and PhoB]. The mutant RNA polymerases did not location of the CRP site relative to the transcription start site respond to transcription activation by activator proteins that (15, 16). Type I promoters are associated with a CRP site that bind upstream of the respective promoters. Transcription by is separated from the basic promoter (-10 and -35 signals) these mutant enzymes was, however, activated in the cases and include the lac P1 and uxuAB promoters. On type II where activators bind to target sites that overlap the promoter promoters, the CRP site is centered around -41/-42 and therefore partially overlaps the basic promoter at the -35 -35 region. Two different mechanisms are proposed for the region. In addition, we examined the responsiveness of the positive control of transcription by activator proteins, one mutant RNA polymerases to transcription stimulation by two requiring the C-terminal domain of the a subunit, and the other activators, PhoB, an activator of the phosphate regu- other not requiring it. lon, and OmpR, which is involved in osmoregulation of the ompF and ompC genes (reviewed in ref. 17). The results Positive control of transcription is one of the common suggest the presence oftwo different mechanisms for positive mechanisms of regulated gene expression in both prokary- control of transcription by activator proteins: one requires otes and eukaryotes. Two mechanisms have been proposed the C-terminal domain of the a subunit, and the other does for this activation, (i) activator-induced conformational not. change of DNA structure around the promoter and (ii) direct contact between activator and RNA polymerase on the DNA MATERIALS AND METHODS (for review see ref. 1). Several mutants ofA repressor protein or cAMP receptor protein (CRP) that still bind to their RNA Polymerases. Wild-type and mutant RNA polymer- respective target sites but are unable to activate transcription ases were assembled in vitro from individually overproduced have been isolated, indicating direct interaction between and purified (3, (3', and (770 subunits and one of the wild-type these activator proteins and RNA polymerase (2-5). or mutant a subunits, as described by Igarashi and Ishihama RNA polymerase of Escherichia coli is composed of four (9). different subunits, a, (3, (3', and one of a variety ofao subunits Transcription Factors. CRP was purified as described (18). (reviewed in ref. 6). One major function of the a subunit is to Purification of OmpR protein and the truncated form of a mediate the assembly ofthe two large subunits, (3and (3', into mutant EnvZll protein, EnvZll*, was described previously the core enzyme structure (7, 8). A set of mutant RNA (19, 20). EnvZll* lacks the N-terminal hydrophobic region. polymerases were constructed that were assembled in vitro Overproduction and purification of PhoB protein and from wild-type ,(, (3', and a,70 subunits and a mutant a subunit PhoR1084, a truncated form of PhoR lacking the N-terminal lacking the C-terminal 73 or 94 amino acid residues (9). The hydrophobic region, were described previously (21, 22). mutant RNA polymerase core enzymes containing C-termi- Template DNAs. All the truncated DNA templates used are nally truncated a subunits exhibited essentially the same illustrated in Fig. 1A. The 240-bp Hpa II fragment containing specific activity of RNA synthesis as the native enzyme. the gal promoter region was isolated from plasmid pBdCl o70 (23). The 344-bp Hinfl fragment containing the pBR-P4 After addition of subunit, these mutant holoenzymes were promoter was isolated from plasmid pBR322 (24). The 516-bp able to initiate transcription accurately from certain promot- HindII1 fragment containing the ompC promoter region was ers. However, they did not respond to transcription activa- obtained from plasmid pUCI-OL (25). The 115-bp BamHI- tion by cAMP/CRP at two type I CRP-dependent promoters HindIII fragment containing the pstS promoter was obtained on the lac and uxuAB genes (9). These results led us to from plasmid pOS1 (26), and its ends were filled-in using the conclude that the C-terminal domain (residues 257-329) ofthe Klenow fragment of DNA polymerase I. The 205-bp EcoRI a subunit is involved in the response of RNA polymerase to fragment containing the lacUV5 promoter was obtained from transcriptional activation by cAMP/CRP. Phenotypes result- plasmid pKB252 (27). ing from known rpoA mutations also suggest that the a Abbreviation: CRP, cAMP receptor protein. The publication costs of this article were defrayed in part by page charge §Present address: Nagoya University, Faculty of Science, Chikusa, payment. This article must therefore be hereby marked "advertisement" Nagoya 464, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 8958 Biochemistry: Igarashi et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8959 A pBR P4 I.: 344 bp S-----lAl-l 241 b gal I- 240 b~p -d 45 b (P1) 6 50 b (P2) ompC 516 bp - 98 b pstS .Z1J 15 bp 41 b B3 ux uAB GAGTGAAATTGTTGTGATGTGGTTAACCCAATTAGAATTCGGGATTGACATGTCTTACCAAAAGGTAGAACTTATACGC CRP site -35 -10 lac P1 AATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA CRP site 35 -10 pBR P4 CGGTATTTTCTCCTTACGCATCTGTiG-CGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTfG CRP site -10 gal P1 TCTTGTGTAAACGATTCCACTAATTTATTTCCATGTCACACTTTTCGCATCTTTGTTATGCTATGGTTATTTCA ompC CRP site "10 TTGCATTTANCAT T TTGAAACATCTATAGCGATAAATGAAACATCTTAAAAGTTTTAGTATCATATTCGTGTTGGATTATTCTGCAT TTTTGGCGAGAAGGACT OrnpR site OmpR site OmpR site .735 - 1 0M pstS TCICTCTGTCATAAAACTGTCATATTCCTTACATATAACTGTCACCTGTTTGTCCTATTTTGCTTCTCG PhoB site PhoB site 0 FIG. 1. Activator-dependent E. coli promoters. (A) Truncated DNA templates used for in vitro transcription. DNA fragments, each carrying a promoter(s) (black areas) and an activator binding site(s) (gray areas), were prepared as described in Materials and Methods. Transcripts from these templates are shown by filled arrows indicating the direction of transcription. bp, Base pairs; b, bases. (B) Nucleotide sequences of the uxuAB, lac, pBR-P4, gal, ompC and pstS promoter regions. Transcription initiation sites are marked by stars. Basic promoter elements (-10 and -35 signals) are underlined by black bars. The binding sites for CRP, OmpR, and PhoB are underlined by gray bars. The nucleotides homologous to the consensus CRP-binding site, OmpR-binding site, and PhoB-binding sites (pho box) are indicated by dashed overlines. The predicted lengths of the run-off transcripts are 45 and mutant RNA polymerases containing a-235 or a-256. Basal 50 nucleotides from the gal P1 and P2 promoters, respec- transcription from P2 in the absence of cAMP/CRP was tively; 241 nucleotides from the pBR-P4; 98 nucleotides from weaker with the mutant enzymes than that with the wild-type the ompC promoter; 41 nucleotides from the pstS promoter; enzymes. When the DNA fragment was preincubated with and 63 nucleotides from the lacUV5 promoter (see Fig. 1). cAMP/CRP, transcription from P1 was markedly stimulated In Vitro Transcription. In vitro transcription from the gal not only for the wild-type but also the mutant enzymes. and pBR-P4 promoters was carried out according to Kajitani Although the basal transcription from P2 was weaker for the and Ishihama (27) with some modifications (9). Transcription mutant enzymes, the level of induced P1 transcription was from the ompC promoter was carried out essentially as almost the same as that by the wild-type enzymes. Inhibition described by Aiba and Mizuno (28), while transcription from ofinitiation at P2 is due to interference with RNA polymerase the pstS promoter was carried out as described by Makino et binding by cAMP/CRP, which attaches to the -35 region of al. (22). RNA products were purified by ethanol precipitation the P2 promoter. and analyzed by electrophoresis in polyacrylamide gels con- Fig. 2B shows transcription from the pBR-P4 promoter. taining 8 M urea. This promoter resembles gal P1 with respect to the location ofthe CRP site relative to the transcription start site (see Fig. 1). Again, transcription from pBR-P4 was stimulated by RESULTS cAMP/CRP with all the enzymes used, although
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