Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum

Abstract

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.

Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants

“…Thus, these proteins require Spo0A based on protein level determination. The finding that Adc requires Spo0A confirms previous studies [34]. Spo0A overexpression affected the abundance of proteins involved in glycolysis, translation, heat shock stress response, and energy production.…”

“…RpoA is a moderately abundant protein. RpoA MW corresponds to that previously reported [34]. In E. coli, Efp stimulates efficient translation and peptide-bond synthesis [3].…”

“…The E. coli and B. subtilis Fba homologs are post-translationally modified [26,32] suggesting that C. acetobutylicum may have another Fba variant, as yet unidentified. RpoA is part of the core RNA polymerase that transcribes genes specified by r factor promoter recognition and thus is important for regulating gene expression [34]. RpoA is a moderately abundant protein.…”

Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum

“…This effect might then be abolished after heat shock by the action of a protein, which opens the mRNA secondary structure and enables RNA polymerase to continue at a higher rate. In this respect, it is of interest that the RNA polymerase from heat-shocked cells of C. acetobutylicum contains an additional protein which reacts to polyclonal antibodies raised against (r2 of E. coli but does not function as a sigma factor (31,32). Experiments are in progress to determine the nature of this protein and its possible function in the expression of C. acetobutylicum heat shock genes.…”

General Biology and Physiology

“…Purified RNA polymerases from C. perfringens and C. acetobutylicum consist of a complex of a, b, b' and s subunits as in other bacteria [347,348], and genes encoding homologues of vegetative and sporulation-specific s factors have been cloned from C. acetobutylicum or identified by hybridization. The sigA (vegetative s A ) gene is expressed as an operon with dnaE, the product of which shows homology to bacterial primases, while the sigE (s E ) and sigG (s G ) genes form a cluster with spoIIGA which encodes a sporulation specific protease [349,350].…”