Fig 3. RNA chaperone activity of TRAM0076. ( A ) TRAM0076 binding to RNA pentaprobes (PP). Twelve 3 0 biotinylated RNA pentaprobes (PP), PP1-PP12, were used as the substrates. Purified recombinant TRAM0076 was added to the binding reaction at indicated concentrations, and RNA-EMSA (rEMSA) was performed to determine binding. Arrows denote the free RNA probes. ( B ) Transcriptional antitermination activity of TRAM0076 determined in E . coli RL211, which carries a cat gene cassette positioned downstream of the trpL terminator (upper panel). Melting of the structured RNA of the trpL terminator allows expression of the chloramphenicol resistance gene cat and growth on plates with chloramphenicol. Strain RL211 transformed with pINIII vector alone or the pINIII carrying MMP0076 , cspE , cspA , and Mpsy_3066 (lower panel). Mid-exponential cultures were induced, diluted 10-fold and spotted on LB plates containing 50 μ g/ml ampicillin and 1 mM IPTG, plus or minus 30 μ g/ml chloramphenicol (+/-Cm). The plates were incubated at 37˚C for 2–3 days. ( C ) Using a FITC fluorescence molecular beacon system (upper panel) to measure the in vitro nucleic-acid melting activity of TRAM0076. Unfolding of the duplex oligodeoxynucleotides relieves quenching and causes an increase in fluorescence intensity. The fluorescence of the heat-denatured molecular beacon (d) is 100%, that of the re-annealed one (a) is background. Addition of 20 μ M (final concentration) of recombinant TRAM0076 protein caused the fluorescence to increase, though less than that caused by the same concentrations of the E . coli CspA and CspE proteins (lower panel).