FIG. 3.
In vitro transcription of the category A Mga-regulated gene emm. Transcription of emm was tested in an in vitro transcription assay with increasing amounts of GAS- and E. coli-purified M6 Mga-His (0, 1.5, and 3.0 μg, lanes 4 to 8) as described in Materials and Methods. DNA template (pKSM416 digested with EcoRI) was incubated with purified σA, core RNA polymerase, Mga (where indicated), and a radiolabeled ribonucleotide mixture. Reaction products were ethanol precipitated and separated on a 6% denaturing polyacrylamide gel. Transcription of the Mga-independent rpsL gene was used as a control in the assay. In vitro transcription was performed on rpsL as described above in the absence and presence of Mga purified from the GAS and from E. coli (lanes 1 to 3). Bands representing the transcribed products of the expected size are indicated by arrows and the names of the promoters. The sizes of the transcripts (in base pairs) were estimated on the basis of the migration of a DNA sequencing ladder.