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 pro-karyotes 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 Echerichia coli enzyme. the Β subunit (Mr = 145000) has a lower molecular weight than the Β subunit (Mr = 1.51000). The Β subunit was identified by its capacity to bind [³H]rifamycin. σ is extremely small (Mr = 44000). y is a peptide chain (Mr = 84000) present once in the Ey monomer. By incomplete dissociation, Β α2, Β'σ and Β'y complexes were obtained. y and 5 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 Mn2+ as bivalent metal ion. Co2+ and Mg2+ 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)] · [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 Mg2+, that is conditions optimal for the E. coli system. Thus, the core and not g 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. [ABSTRACT FROM AUTHOR]