Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription
Figure 2
(a) Effect of D2O on cell-free transcription. First mRNA was transcribed in a cell-free system with 0%, 20%, 40%, and 60% D2O, respectively. Then the mRNA was purified and cDNA was produced using a reverse transcriptase. The amount of cDNA in each sample was subsequently measured by real-time PCR. At 60% D2O, the mRNA yield was increased by about a factor of two to three compared to samples with H2O. (b) Relative transcription rate in the presence of D2O. (c) The effect of D2O on GFP translation in a cell-free system. mRNA was transcribed and purified and in a second step used as template in a cell-free system with different D2O concentrations. (d) Relative GFP synthesis rate in the presence of D2O. (e) Effect of D2O on the folding time of GFP. mRNA translation in the cell-free system was stopped by adding the antibiotic chloramphenicol to the sample at the time point (ribosome deactivation). Any increase in fluorescence after is due to maturation of already translated GFP. The maturation time is remarkably longer in D2O (66% increase of at a concentration of 60%) than in H2O.
