FIG. 5.
Comparison of His6-tagged and untagged PrfA proteins and the effect of a His6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfALm (P), 230 nM nontagged PrfALm (Pnt), 12 nM PrfA*Lm (P*), and 12 nM nontagged PrfA*Lm (P*nt) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes PhlyLm (A) and PactALm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments PhlyLm (B) and PactALm (D). The mRNA was labeled with [α-32P]CTP during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.