Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

Abstract

The role of the a subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated a subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the a subunit, and the other not requiring it.Positive control of transcription is one of the common mechanisms of regulated gene expression in both prokaryotes and eukaryotes. Two mechanisms have been proposed for this activation, (i) lacking the C-terminal 73 or 94 amino acid residues (9). The mutant RNA polymerase core enzymes containing C-terminally truncated a subunits exhibited essentially the same specific activity of RNA synthesis as the native enzyme. After addition of o70 subunit, these mutant holoenzymes were able to initiate transcription accurately from certain promoters. However, they did not respond to transcription activation by cAMP/CRP at two type I CRP-dependent promoters on the lac and uxuAB genes (9). These results led us to conclude that the C-terminal domain (residues 257-329) ofthe a subunit is involved in the response of RNA polymerase to transcriptional activation by cAMP/CRP. Phenotypes resulting from known rpoA mutations also suggest that the a subunit is involved in positive control by certain transcription activators (10-14).In the present study, we extended this line of experiments to type II CRP-dependent promoters. Most CRP-dependent promoters can be classified into two types depending on the location of the CRP site relative to the transcription start site (15, 16). Type I promoters are associated with a CRP site that is separated from the basic promoter (-10 and -35 signals) and include the lac P1 and uxuAB promoters. On type II promoters, the CRP site is centered around -41/-42 and therefore partially overlaps the basic promoter at the -35 region. In addition, we examined the responsiveness of the mutant RNA polymerases to transcription stimulation by two other activators, PhoB, an activator of the phosphate regulon, and OmpR, which is involved in osmoregulation of the ompF and ompC genes (reviewed in ref. 17). The results suggest the presence oftwo different mechanisms for positive control of transcription by activator proteins: one requires the C-terminal domain of the a subunit, and the other does not. MATERIALS AND METHODSRNA Polymerases. Wild-type and mutant RNA polymerases were assembled in vitro from individually overproduced and purified (3, (3', and (770 subunits and one of the wild-type or mutant a subunits, as described by Igarashi and...

A nested set of C‐terminal deletions of the α subunit of Escherichia coli RNA polymerase define regions concerned with assembly, proteolysis, stabilization and transcriptional activation in vivo

“…The contact site on a for the CRP¹cAMP complex at the class I promoter lacP1 has been pinpointed to residues 265¹269 (N-terminal proximal portion of H1) (Zou et al 1992;Murakami et al 1996);and Sharif et al (1994) have shown that a mutation at A272 (H1) increases lacZ expression. That truncated a derivatives retaining these residues (a 271þ2 ¹a 316þ3 ) fail to respond to CRP¹cAMP (as has been shown previously for a 235 and a 256 in vitro Igarashi et al 1991b;Kolb et al 1993)) indicates sequences beyond the contact site play a role in activation, presumably by maintaining the correct spatial orientation and/or protecting the a C-terminal domain from proteolysis. An alternative possibility, that sequences beyond H1 interfere with activation by CRP¹cAMP unless correctly positioned, is unlikely given their rapid removal.…”

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

“…The ␣ subunit, consisting of 329 amino acid residues, is composed of two structural domains, each responsible for distinct functions (1-3) and each forming independent structural domains connected by a protease-sensitive flexible linker (4-6). The amino (N)-terminal domain from residues 20 to 235 plays a key role in RNA polymerase assembly by providing the contact surface for ␣ dimerization and binding of ␤ and ␤Ј subunits (7-10), whereas the CTD from residues 235 to 329 plays a regulatory role by providing the contact surfaces for trans-acting protein factors and cis-acting DNA elements (11)(12)(13)(14).Whereas the regulation of many E. coli promoters involves a single factor, some promoters are regulated by two or more transcription factors, and such coregulation systems involving multiple species of transcription factors can couple gene expression to diverse environmental conditions. Knowledge of the molecular mechanism of prokaryotic transcription regulation involving more than two factors would contribute much to understanding of the events carried out in eukaryotes, because the regulation of gene transcription in eukaryotes generally involves the action of multiple transcription factors.…”

“…The ␣ subunit, consisting of 329 amino acid residues, is composed of two structural domains, each responsible for distinct functions (1)(2)(3) and each forming independent structural domains connected by a protease-sensitive flexible linker (4)(5)(6). The amino (N)-terminal domain from residues 20 to 235 plays a key role in RNA polymerase assembly by providing the contact surface for ␣ dimerization and binding of ␤ and ␤Ј subunits (7)(8)(9)(10), whereas the CTD from residues 235 to 329 plays a regulatory role by providing the contact surfaces for trans-acting protein factors and cis-acting DNA elements (11)(12)(13)(14).…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…In addition to the RNAPH s 4 domain, the interactions between the C-terminal domain of the a-subunit of RNAPH and OmpR or some other transcription activators were described [31][32][33][34][35] . To elucidate the initiation steps for transcription activation by PmrA, the interactions between PmrA-DNA and the s 4 or C-terminal domain of the a-subunit of RNAPH remain to be characterized, which is in progress.…”