The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system
Fig 7
CsrA acts as a positive regulator for Glyceraldehyde 3-phosphate (Gap) by preventing premature transcriptional termination of the PPP/Glycolysis-operon.
A) RNA secondary structure Mfold-prediction of the CsrA-binding region inside the gap gene reveals two major conformations: the left one contains a potential hairpin-terminator while the A(N)GGA-motif is covered in a double-strand region with low affinity to CsrA. The right one, shows the A(N)GGA-motif located in an open loop with high CsrA-interaction affinity and the hairpin structure is disrupted. Below, the nucleic acid sequence is shown that was used for Mfold modeling and the transcription termination assays. Red, CsrA-binding site A(N)GGA; green, the potential transcription terminator hairpin; blue, the putative auxiliary element of Rho-dependent termination. B) Left panel: In vitro transcription termination assay in presence of 1 μM of purified NusG-protein and varying concentration of Rho- and CsrA-protein (+ 0.5 μM, ++ 1μM; Lane 1: no Rho, no CsrA, lane 2: 1 μM Rho, no CsrA, lane 3: 1 μM Rho, 0.5 μM CsrA, lane 4: 1 μM Rho, 1 μM CsrA, lane 5: 1 μM Rho, no CsrA, lane 6: 1 μM Rho, 1 μM CsrA). A representative 10% urea-PAGE gel shows the formation of the truncated transcript from the Rho-dependent termination without CsrA and the full-length transcript with CsrA. Right panel: In vitro transcribed of the run-off fragment and the marker showing the size of the fragment. C) Regulatory model of the transcription of the PPP/Glycolysis operon. In absence of CsrA, Rho-dependent termination within the operon is responsible for polarity effects downstream of the transcriptional block. This leads to reduced transcript-levels of the gap gene whereas the tkt gene is not affected. When CsrA binds to the RNA, an anti-terminator structure is favored preventing that the elongation complex stalls at the hairpin structure. As a consequence, only the presence of CsrA ensures the efficient transcription of the glycolysis/gluconeogenesis genes of the operon.