Figure 3
MBP::AutA and MBP::AutR directly binding the PupaB promoter region and altering the upaB transcription in vitro.
(A) Non-radioactive EMSA detecting the band shift of PupaB promoter region. PupaB DNA fragment (200 bp) and the negative control DNA fragment (200bp for upaB coding region) were amplified by PCR and used as EMSA probes. EMSAs were conducted by adding increasing amounts of MBP::AutA (up panel) and MBP::AutA (down panel) fusion proteins in each reaction mixture. The purified His-MBP fusion protein acted as the negative control for EMSA tests. The gels were stained with 1× SYBR Gold nucleic acid solution. (B) In vitro transcription assays to determine the effect of AutA and AutR on upaB transcription. The amount of RT-PCR products for both PupaB-upaB (upper panel) and Ptac-malE (lower panel) (control) with or without adding MBP::AutA and MBP::AutA fusion proteins in each reaction mixture. The RT-PCR product directly corresponds to the transcription level of the corresponding RNA synthesized in vitro.
