Figure 4.

Csp proteins cause transcription antitermination at the metY-rpsO operon in vitro and in vivo. (A) A DNA fragment containing two consecutive intrinsic terminators, t₁ and t₂, found downstream of metY and genetically fused to the T7 A1 promoter, was used as a template to examine the effect of CspE on the termination at these terminators. The experiment was performed and data analyzed as described in the Fig. 1 legend. The combined RE values at t₁ and t₂ were calculated (RE = {[RO transcripts]/[total transcripts (t₁+ t₂₊ RO)]} × 100%) and are graphically presented (Right). (B) E. coli cells were transformed with the pINCspA, pINCspC, or pINCspE plasmids, grown at 37°C to OD₆₀₀ of 0.3–0.4, and csp expression was induced by the addition of 1 mM IPTG. After 60 min incubation at 37°C, half of the culture was subjected to the isolation of total RNA and Northern blot analysis by using the metY gene as a probe (lanes 1–6), whereas the other half was analyzed by SDS/PAGE on a 17.5% gel (lanes 1′-6′). The overexpressed proteins are indicated by black dots. (C) RL211 E. coli cells containing a cat gene cassette positioned downstream of the trpL terminator (36) were transformed with pINCspA, pINCspC, or control pINIII, and spotted on plates containing 30 μg/ml chloramphenicol and1 mM IPTG. The results of overnight cell growth are presented.