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Transcription factors are one of the most important components in transcription regulation, given their ability to sense environmental fluctuations and modulate gene expression accordingly (Babu and Techmann 2003; Browning and Busby 2016). These proteins usually have two domains, where one receives internal/external signal, while the other interacts with the DNA (Babu and Teichmann 2003).

Upon binding to a promoter, a transcription factor may limit the interaction of the RNAp with the promoter, thus hampering transcription. These are known as repressors. Several mechanisms have been identified by which they are able to halt transcription (Figure 2.8A). For some promoters this occurs by steric hindrance, where the operator site overlaps with the -10 and -35 elements recog- nized by the RNAp, thus the binding of the repressor to the operator site blocks the RNAp access to the promoter region (Browning and Busby 2004). Repression can also be achieved by DNA looping, where repressor molecules bind simultaneously to operator sites located upstream and downstream of the promoter region inducing the formation of a DNA loop, also preventing the RNAp to access the promoter region (Schleif 2010; Browning and Busby 2016). For other pro- moters, the repressor can act as an “anti-activator”, thus preventing activator molecules from “ac- tivating transcription” (Browning and Busby 2016).

Additionally, transcription factors can also actively recruit RNAp molecules to the promoter re- gion, thus activating transcription. Here, they are called activators and there are several mecha- nisms by which they can activate the transcription process (Lee et al. 2012) (Figure 2.8B). In Class I activation, the activator binds to an operator located upstream of the promoter region, and recruits the RNAp by interacting with the C-terminal domain of its α-subunit (Busby and Ebright 1999; Lee et al. 2012). In Class II activation, the operator region where the activator binds overlaps with the -35 element, thus the activator recruits the RNAp by interacting with the domain 4 of the sigma factor (Lee et al. 2012; Browning and Busby 2016). Transcription activation can also occur through conformational changes on the promoter DNA. Here, the activator binds to the operator site located at or near the -35 and -10 elements, and rearrange them, so that they are better posi- tioned for the binding of the RNAp (Lee et al. 2012; Browning and Busby 2016).

Given that the interaction between transcription factors and promoter is dependent on the promoter sequence and the regulatory protein structure (Babu and Teichman 2003; Browning and Busby 2004, 2016), this type of mechanisms allows the cells to diversify their gene expression profile. Some transcription factors can be affected by specific molecules called inducers. For instance, these molecules can inactivate repressors, thus promoting transcription. One of the best known examples is the repressor of the lac operon, LacI, which is inactivated by its inducer, lactose (Jacob and Monod 1961). Other inducers can promote gene expression by increasing the functionality of its activators or by inverting the repressor function, turning it into an activator, as in the case of AraC, the repressor of the Arabinose operon (Englesberg et al. 1965; Schleif 2010).

Figure 2.8: Examples of repression and activation at promoters by transcription factors. Represented are several mechanisms by which these transcription factors are able to repress (A) and activate (B) tran- scription. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Microbiology] (Browning and Busby 2016), copyright (2016).

Several gene expression inducers are not synthesized by the cell, but rather enter the cell from the environment. For this to occur, these molecules must travel through the cell membrane. The E.

coli’s membrane (Figure 2.9) consists of two layers, the outer and the inner membrane, separated

by the periplasmic space (Zimmermann and Rosselet 1977; Alberts et al. 2002). The outer mem- brane is semi-permeable, allowing the crossing of lipophilic, and some small uncharged molecules (Finkelstein 1976; Willey et al. 2008), while preventing larger and ionic molecules from crossing (Decad and Nikaido 1976). On the outer membrane, transmembrane proteins, known as porins, act as channels for the entrance/exit of some specific molecules (Alberts et al. 2002; Nikaido 2003; Willey et al. 2008). Along with the lipid bilayers, these proteins are important for the dynamics of intracellular level of inducer molecules, and consequently the gene expression response to their presence in the environment.

The lactose intake system is one of the most studied mechanism for inducer intake (Jensen et al. 1993; Marbach and Bettenbrock 2012). The majority of these studies were conducted in a regime of low concentrations of lactose, where the rate of the target gene production presents an almost linear dependence on the intracellular level of inducer (Jensen et al. 1993).

Figure 2.9: An illustration of E. coli’s membrane. The membrane consists of two lipid bilayers, the outer and the inner membrane, which are separated by the periplasmic space and peptidoglycan. Bound to the membrane are porins and other transmembrane proteins. Reproduced from (Willey et al. 2008), with per- mission from McGraw-Hill Education, copyright (2008).

At these concentrations (less than 0.25 mM), it was found that the cellular intake of lactose had a positive feedback, due to the activity of the lactose permease, LacY (Jensen et al. 1993; Ozbudak

et al. 2004; Marbach and Bettenbrock 2012). This is a proton symporter protein, that uses the

transmembrane proton gradient to simultaneously transport protons and lactose (Ramos and Kaback 1977; Kaback 1983). Upon the entrance of lactose molecules in the cell, the lactose operon is activated (see section 2.3.2 for details), producing LacY proteins, which in turn lead to an in- crease in the intake of lactose molecules (Jensen et al. 1993; Ozbudak et al. 2004). At higher concentrations of lactose, the role of LacY is no longer significant, with the lactose molecules entering the cell through alternative symporters and potentially through passive diffusion (Decad and Nikaido 1976; Jensen et al. 1993).

At high concentrations, the kinetics of inducer intake is less studied, mainly because under this condition, the activity of the target gene is close to full induction, which no longer reflects the intracellular changes in the inducers level. In Publication I, to address this issue we implemented a method to characterize the intake of IPTG in cells lacking the LacY protein, using in vivo single RNA measurements, at the single RNA and single cell level (Golding et al. 2005).

Another factor that can influence the intake of inducer molecules is temperature fluctuations, which is known to affect several cellular processes in E. coli (Yamanaka 1999; Arsène et al. 2000).

For instance, it can alter the functionality of proteins (Arsène et al. 2000) and also change the physical properties of the cell membrane and cytoplasm (Sinensky 1974; Yamanaka 1999; Oliveira

et al. 2016b). Changes in these properties are expected to have severe effects on the intake kinetics

of inducers. To address this, in Publication IV we assessed the temperature dependence of the intake process of the inducer IPTG at different temperatures using in vivo single RNA measure- ments techniques.