Broccoli aptamer allows quantitative transcription regulation studies in vitro

Abstract

Quantitative transcription regulation studies in vivo and in vitro often make use of reporter proteins. Here we show that using Broccoli aptamers, quantitative study of transcription in various regulatory scenarios is possible without a translational step. To explore the method we studied several regulatory scenarios that we analyzed using thermodynamic occupancy-based models, and found excellent agreement with previous studies. In the next step we show that non-coding DNA can have a dramatic effect on the level of transcription, similar to the influence of the lac repressor with a strong affinity to operator sites. Finally, we point out the limitations of the method in terms of delay times coupled to the folding of the aptamer. We conclude that the Broccoli aptamer is suitable for quantitative transcription measurements.

Fig 1. In vitro transcription method and theory.

A: Schematic representation of the method. The highlighted part of the transcription curve represents the linear part of the data that is used to determine the transcription rate. B: Various regulational states and their corresponding statistical weights.

Fig 2. Transcription rates scaled to promoter occupation.

A: Fluorescence increase for transcription experiments at RNAP concentrations of 10 nM, 100 nM, 333 nM and 3.33 μM, from low to high rates. The linear part used to determine the transcription rate is shown in green. B: The transcription rates corresponding to different concentrations of T7 RNA polymerase (pink dots), scaled to the theoretical promoter occupancy (black line). The RNAP binding free energy as found by a linear fit to the data is ϵ_p,_s = −18.9 ± 0.7 k_BT.

Fig 3. Repression using the Lac operon.

A: Fold change for various repressor concentrations. The solid lines are the predicted fold changes for the different concentrations of RNAP with ϵ_r,s = −23.6k_BT ± 0.3 k_BT. The blue line shows the fold change in case of dimers, which follows from a binding energy that is ln(2) lower than the energy used for tetramers. B: Data from A collapsed onto a master curve.

Fig 4. Nonspecific DNA acts as a repressor.

Master curve for three different RNAP concentrations, with the addition of nonspecific DNA (salmon sperm).