title: The RNA polymerase alpha 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 alpha 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 (alpha)-rplQ (L17) found in the alpha-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti alpha 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 alpha and E. coli beta, beta', and sigma subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti alpha functions are conserved in heterologous host E. coli even though the two alpha 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.
authors.affiliation: Department of Biological Sciences, Stanford University, California 94305, USA.
authors.forename: Melicent C
authors.initials: MC
authors.lastname: Peck
authors.forename: Tamas
authors.initials: T
authors.lastname: Gaal
authors.forename: Robert F
authors.initials: RF
authors.lastname: Fisher
authors.forename: Richard L
authors.initials: RL
authors.lastname: Gourse
authors.forename: Sharon R
authors.initials: SR
authors.lastname: Long
mesh_terms: D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D021122:Protein Subunits; D016962:Sinorhizobium meliloti / Q000201:enzymology*; D015533:Transcriptional Activation
chemical_list: D021122:Protein Subunits; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit