Boireau et al. report, the first measurements of RNA kinetics in individual cells show an unaccounted for two and a half minutes in the protein's schedule.
Using groups of cells, researchers have previously determined averages for how many nucleotides RNA polymerase II can add in a minute (900–1,800), its initiation rate, and other variables. To refine these figures, Boireau et al. gauged how fast single cells transcribe a segment of the HIV genome. The researchers altered the segment to contain multiple binding sites for the protein MS2. By adding GFP-labeled MS2, which binds to the transcript, the researchers could track the growth of the new RNA strand. Mathematical models then allowed the team to calculate the timing of different steps.
RNA polymerase II added more than 1,800 bases/min—above the average gleaned from in vitro studies, suggesting that an HIV protein might boost its speed. The polymerase spent just over five and a half minutes at the transcription site. Of this time, making the RNA strand took 114 seconds, and processing the 3′ end and other housekeeping tasks required another 63 seconds. That left 156 seconds unexplained.
What the protein does during this gap is unclear. The extra time might indicate that the protein pauses on the promoter before it starts transcribing or that it performs some other undiscovered function. The next step, the researchers say, is making the same measurements for cellular genes.