Transcription in Lactobacillaceae

This paper describes the isolation and the properties of DNA‐dependent RNA polymerase from Lactobacillus curvatus. The enzyme is highly labile and shows a tendency to dissociate into fragments. Therefore, a special purification procedure also suitable for the isolation of labile RNA polymerases from other prokaryotes was developed. Three enzyme species, E (core enzyme), Eσ (full enzyme) and Ey were obtained. The subunit composition corresponds to that of other prokaryote RNA polymerases. In contrast to Escherichia coli enzyme, the β′ subunit ( M r = 145000) has a lower molecular weight than the β subunit ( M r = 151000). The β subunit was identified by its capacity to bind [ 3 H]rifamycin. σ is extremely small ( M r = 44000). y is a peptide chain ( M r = 84000) present once in the Ey monomer. By incomplete dissociation, βα 2 , β′σ and β′y complexes were obtained. y and σ have never been found together in the same enzyme particle. In contrast to RNA polymerase from E. coli , Eσ from L. curvatus exhibits optimal activity in the presence of Mn 2+ as bivalent metal ion. Co 2+ and Mg 2+ also activate though with considerably lower efficiency. All subunits except y were isolated in pure state. σ was catalytically active. y could only be obtained as a complex with β'. On single‐stranded DNA, Eσ and E are equally active. For the transcription of double‐stranded DNA, σ is absolutely required. Even the transcription of poly[d(A‐T)] σ [d(A‐T)] is strongly stimulated by σ. σ from L. curvatus is able to replace σ from E. coli on E. coli core enzyme even for the formation of the stable preinitiation complex. This effect requires Mg 2+ , that is conditions optimal for the E. coli system. Thus, the core and not σ appears to determine the requirement for the bivalent metal ion. Different double‐stranded templates are transcribed with highly different efficiency. The ionic strength optimum is different for different templates. Ey in contrast to E exhibits a low, though significant background activity on double‐stranded DNA. It is stimulated by σ to twice the specific activity of Eσ. Thus, σ and y act synergistically though they appear to exclude each other on the core.