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

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 α 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 (α)- rplQ (L17) found in the α-proteobacteria. In vivo, S . meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA , demonstrating that S. meliloti α 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 α and E. coli β, β′, and σ 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 α functions are conserved in heterologous host E. coli even though the two α 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.