Fig. 2.

Activities of the R. sphaeroides rRNA promoters in vitro with or without purified R. sphaeroides CarD. (A) In vitro transcription of the R. sphaeroides rrnB promoter with 20 nM R. sphaeroides RNAP with or without CarD_Rsp (wedge indicates CarD_Rsp range of 5 to 2,560 nM) in buffer with 170 mM NaCl. (B) Fold activation of the R. sphaeroides rrnB promoter by CarD_Rsp (with/without the indicated concentration of CarD_Rsp) from experiments like that in A. The RNA I transcript is from a plasmid-encoded promoter. Error bars indicate SD from n = 3 separate assays. (C) In vitro transcription of the R. sphaeroides rrnA or rrnC promoters with R. sphaeroides RNAP as in A with or without 1,280 nM CarD or of the R. sphaeroides rrnB promoter with 10 nM E. coli RNAP with or without 20, 40, or 80 nM CarD. (D) Fold activation of the R. sphaeroides rrnA, rrnB, and rrnC promoters by 1,280 nM CarD with SD from n = 3 assays for each promoter. (E) Fold activation of the R. sphaeroides rrnB promoter by RNAP_Rsp and 1,280 nM wild-type CarD or W91A, W91L, or Q31A/I33A/R53A triple-mutant CarD (SD from n = 3 assays). Wild-type CarD used in B and E was from different protein preparations, so differences in specific activities could account for the slight differences in fold activation.