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

Abstract

Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the ␣ subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (␣)-rplQ (L17) found in the ␣-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti ␣ supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti ␣ and E. coli ␤, ␤, and subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) elementand Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti ␣ functions are conserved in heterologous host E. coli even though the two ␣ subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.The ␣-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 ␣ 2 ␤␤Ј core subunit structure found in most bacteria (23,45). In addition 70 , 54 , and 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 t...

A recombinantMycobacterium tuberculosis in vitrotranscription system

“…The procedure is fast, does not require biosafety containment facilities and yields a σ factor‐free preparation. Moreover, hybrid RNAP can easily be generated, and mutations that could otherwise be lethal or could severely impair bacterial growth can be introduced in any of the proteins (Tang et al , 1995; Peck et al , 2002). This work describes the preparation and the use of a recombinant in vitro transcription system for the study of gene expression regulation in M. tuberculosis .…”

Similarity and divergence between the RNA polymerase α subunits from hyperthermophilic Thermotoga maritima and mesophilic Escherichia coli bacteria

“…2A). Slower migration in gel was also observed with the α subunit of Sinorhizobium meliloti RNAP (Peck et al, 2002).…”

Rhodobacter capsulatus nifA1 Promoter: High-GC −10 Regions in High-GC Bacteria and the Basis for Their Transcription

“…This method uses the six-histidine tag on the R. capsulatus ␣ subunit, facilitating RNAP purification when crudely prepared R. capsulatus ␤ and ␤Ј subunits overexpressed in E. coli are used. While our study was in progress, this method was shown to work efficiently with the ␣ subunits from other ␣-proteobacterial organisms for in vitro transcription with the E. coli ␤ and ␤Ј subunits (17,23). The R. capsulatus ␣ subunit (45% identical to the E. coli ␣ subunit) is overexpressed as an approximately 43-kDa subunit and purified by using an Ni 2ϩ affinity column with urea.…”

“…Kranz et al previously noted that R. capsulatus and other ␣-proteobacteria (see reference 15 for a review) may have Ϫ10 features different from those of organisms with a lower GϩC content (3,4). For another ␣-proteobacterium, Sinorhizobium meliloti, most of the 70 promoters that have been characterized are not transcribed by the E. coli RNAP in vivo or in vitro, but the S. meliloti RNAP can initiate transcription at typical E. coli 70 promoters (23). The ␣-proteobacterium Caulobacter crescentus 73 RNAP recognizes E. coli 70 promoters lacUV5 and neo, whereas E. coli does not recognize typical C. crescentus 73 promoters (28).…”