The RNA Polymerase α Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro

The α-proteobacterium Sinorhizobium meliloti is able to live either as a soil saprophyte or in a symbiotic relationship with members of the legume family, such as alfalfa. Recent studies have focused on understanding how rhizobia adapt to these unique environments, especially at the level of gene expression (6, 13, 62). For the symbiosis to occur, expression of a subset of genes, such as the nod and nif genes, must be tightly regulated (reviewed in reference 22). As is the case with other bacteria, much of the gene regulation occurs at the level of initiation of transcription (28). To facilitate our studies of transcription and its regulation in S. meliloti, we must understand RNA polymerase (RNAP) structure and function. Previous work demonstrated that RNAP from S. meliloti displays the characteristic α2ββ′ core subunit structure found in most bacteria (23, 45). In addition σ70, σ54, and σ 72 homologs have been cloned from S. meliloti (47, 48, 52, 55). These results are consistent with the evidence that bacterial RNAPs display overall sequence and functional similarities, although they can exhibit some differences in individual steps during transcription such as promoter recognition and promoter escape (4). Since only a limited number of S. meliloti promoters have been characterized, the cis-acting elements are not yet as well defined as in Escherichia coli promoters (7, 23, 55). Nevertheless, S. meliloti RNAP can initiate transcription at typical E. coli promoters (19, 23). However, most S. meliloti promoters that have been characterized are not transcribed by E. coli RNAP in vivo or in vitro (5, 19), perhaps because the S. meliloti Eσ70 homolog recognizes these promoters slightly differently from E. coli Eσ70 or because these promoters utilize σ factors or transcription activators not found in E. coli. In the past decade, based primarily on work with E. coli, RNAP α has emerged as a key player in both basal transcription and in transcriptional activation (reviewed in references 20 and 29). RNAP α consists of two independently folded domains connected by a flexible linker (9, 64). The amino-terminal domain (αNTD) is required for α dimerization, for RNAP assembly, and for interaction with a subset of transcription factors (32, 51); the carboxy-terminal domain (αCTD) is required for binding to the upstream (UP) element, an A+T-rich sequence found upstream of the −35 hexamer, and for interaction with a number of transcription factors (35, 53). Several screens for α mutants have identified residues required for the activation of transcription. These α-activator contacts help recruit RNAP to promoters and/or stimulate the isomerization of the RNAP-promoter complex from the closed to the open state (46). Furthermore, the αCTD may also interact with the σCTD during transcription initiation at some promoters (29). In some cases, α-activator contacts appear be species specific, suggesting that α and activators have coevolved. For example, Agrobacterium tumefaciens α is required for VirG-activated transcription of the virB promoter in E. coli (39). Similarly, transcription activation from the Bacillus subtilis phage A3 promoter requires RNAP containing B. subtilis α and is not supported by E. coli RNAP (43). In both cases, the species specificity of the α-activator contact was mapped to the αCTD (40, 43). Interestingly, Bordetella pertussis α reconstituted into E. coli RNAP does not support transcription at the E. coli CAP-dependent lac promoter (58), suggesting that different activator-α specificities may exist, despite striking sequence homologies in the αCTDs of B. pertussis and E. coli. Our ultimate goal is to understand how α interacts with transcription factors to initiate transcription at S. meliloti promoters. In this paper we describe the cloning and characterization of the S. meliloti RNAP α subunit. Furthermore, we establish that S. meliloti α can functionally replace E. coli α in vivo and that S. meliloti α reconstituted into E. coli RNAP holoenzyme can support both UP element- and Fis-dependent transcription in vitro. These results suggest that the study of transcription activation in S. meliloti may be facilitated by utilizing tools developed for E. coli RNAP.