There are several techniques that can be used to measure promoter strength, such as reporter gene assays and electrophoretic mobility shift assays (EMSA). Promoter strength can be estimated by measuring any reporter gene product activity, such as the
lacZ gene product (-galactosidase) enzyme activity, assuming that this activity reflects
promoter strength. The principle of the -galactosidase assay is to measure the hydrolysis of O-nitrophenyl-β-D-galactopyranoside (ONPG). ONPG is hydrolyzed by water giving galactose (colourless) and O-nitrophenol (yellow). The amount of O-nitrophenol formed is normally proportional to the amount of -galactosidase and the reaction time. The amount of O-nitrophenol produced can be measured by determining the absorbance at 420 nm. At the end of the assay, the reaction is stopped by adding Na2CO3, leading to the rapid increase in the reaction mixture pH to around 11. At this pH, a certain amount of the O-nitrophenol is converted to the yellow-coloured anionic form and -galactosidase is inactivated.
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EMSA is a technique mainly used to study protein-RNA or protein-DNA interactions. EMSA can assess binding complexes and determine if a protein is capable of binding to a given DNA or RNA sequence. This assay depends on the electrophoretic separation of protein-RNA or protein-DNA mixtures on an agarose or polyacrylamide gel depending on their sizes. Therefore, if the protein is capable of binding to DNA or RNA, the resulting complex will move slowly through the gel and demonstrate a band with a larger size than a band of protein alone. This technique can be applied to assess promoter strength by adding different concentrations of RNAP (protein) with the promoter. Strong promoters will produce binding complexes (promoter-RNAP complexes) with low RNAP concentrations. However, weak promoters will need higher RNAP concentrations to produce complexes.
Several previous studies have tried to indirectly measure promoter strength and RNAP flux through given genes in living cells. Kelly and colleagues (2009) tried to indirectly measure promoter strength by fusing them to genes coding for green fluorescent protein (GFP) and calculate promoter strength depending on the amount of GFP produced by these genes under the control of different promoters. Using their calculations, which involved estimates of the rate of GFP synthesis, folding and maturation, and RNA half-life, they were able to measure polymerase per second (PoPS) for their promoters. Another recent study used computational methods to design genetic circuits, which then inserted in different specific locations on the DNA and transformed into bacterial strain. They were able to indirectly calculate the amount of RNAP per second per the standard promoter used in their study (Nielsen et al., 2016). To date, to my knowledge, no reliable technique has been developed to directly measure promoter activity in vivo. The two aforementioned techniques and many other
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techniques rely solely on measuring genes’ final products. The reporter gene assay measures the amount of final product produced by a gene, which can be compared to another gene’s products to evaluate which gene is stronger. The EMSA technique depends on measuring the sizes of protein-DNA or protein-RNA complexes in vitro and changing the protein concentrations to determine which protein concentration produces binding complexes. Hence, both techniques are indirect and in vitro.
Considering a lack of reliable methods for measuring promoter activity directly in real-time, as inside the cell, an alternative technique for measuring promoter activity is desirable. In this study, I developed a new, real-time method to directly assess promoter strength depending on RNAP activity. This method allowed us to yield more measurements regarding promoter strength, such as the promoter competitivity, promoter occupancy index (POI), promoter escape index (EI), fragment occupancy percentage (FOP) and PoPS.
All samples were tested using a chromatin immunoprecipitation (ChIP) experiment to immunoprecipitate DNA fragments that were bound to RNAP in each sample. The resultant DNA fragments were quantified by quantitative polymerase chain reaction (qPCR) using specific primers. qPCR employs the same procedural steps as conventional PCR. However, in qPCR, as the DNA is labelled with a fluorescence dye, the amount of the amplified DNA can be accurately counted by monitoring the fluorescence emitted in each thermal cycle. At the end of each qPCR run, readings are given as cycle threshold (Ct), which describes how many cycles emitted a fluorescence exceeding the background signal. The Ct value is relative to the initial amount of the DNA, in that fluorescence will be detected earlier in samples with high initial DNA concentrations (i.e., samples with high DNA concentrations will yield low Ct values). After qPCR, these Ct values were used
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to calculate the amount of DNA fragments of interest that were occupied by RNAP in each sample. These experiments were performed in triplicate for each sample as a biological repeat. For each biological repeat, qPCR was performed in triplicate as a technical repeat.
This new direct method developed in this study was applied on two different systems: the lac operon on the chromosome and the lac operon in pRW50 with different promoters. For the lac operon on the chromosome system, the lac operon activity was tested on the chromosome twice: one time before and after rifampicin treatment and another time before and after IPTG treatment. In qPCR, seven newly designed primer pairs were used. A primer pair for the promoter region (lac0), five sets of primer pairs distributed along the lac operon (lac1, lac2, lac3, lac4, and lac5) and a primer pair for a control region. For the lac operon in pRW50, nine synthetic promoters with different activities inserted into the pRW50 plasmid, just before the lac operon, were tested with and without rifampicin treatment. Then, qPCR was applied using four sets of primer pairs to quantify how many RNAPs were attached to the promoter, two downstream regions on the lacZ gene and a control region.
The Cts obtained from all qPCR tests were used to calculate the occupancy value of each region. These occupancy values were then used to calculate how competitive is the promoter in recruiting RNAPs, how efficiently the RNAP recruited by the promoter (i.e., POI), how efficiently the promoter allows RNAP to escape (i.e., EI), the time interval between RNAPs transcribing downstream regions (Tint), downstream fragments occupancy percentage (FOP) and the rate at which RNA polymerase moves past a given position on the DNA (i.e., PoPS).
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