INTRODUCTION
1.4 The diversity of transcriptional regulatory mechanisms
There is great diversity in bacterial regulatory architectures, often representing sophisticated control systems that respond sensitively to environmental changes [23, 26–28]. At a basic level, we can think of regulatory architectures as arrangements of transcription factors that interact with one another and with RNAP. Figure 1.6 shows the diversity of known regulatory architectures in E. coli as recorded in RegulonDB. Many of the promoters in RegulonDB have no regulatory annotations, which indicates either that the promoters are constitutive or their architectures have not yet been identified. Among promoters with regulatory annotations, we see that it is most common for a promoter to have a single binding site annotation, but there remain many promoters with two or three recorded transcription factor binding sites.
The data for promoters with four or more binding sites are not included in this plot.
The distribution of transcription factor binding sites can give us a sense of how a promoter is regulated, but there are a number core regulatory mechanisms that cannot be captured by mapping binding sites alone. In Figure 1.7 we use the classic example of thelacZYAoperon to illustrate three important regulatory mechanisms that go beyond simple transcription factor binding. These mechanisms are allostery, looping, and binding by architectural proteins.
Allostery
Allostery is an exceedingly common phenomenon in both eukaryotes and prokary- otes, in multiple classes of proteins. As discussed in detail in Chapter 2, an allosteric protein switches between two or more conformations which can have different prop- erties. Binding of a ligand to the protein can dramatically increase the probability that the protein will adopt a particular conformation. For transcription factors, this means that allostery serves as a form of “one-component signaling,” where a small molecule signal directly stimulates a response such as a change in gene regulation [23].
Both CRP and LacI are allosteric transcription factors. In the case of the global regulator CRP, the small molecule cAMP must be present in order for CRP to adopt a conformation that binds to DNA with high affinity. Conversely, when the small molecule allolactose (a derivative of lactose) binds to LacI, LacI adopts a conformation that has a weak affinity for DNA. Because LacI acts as a repressor for the lacZYA operon, this means that allolactose acts as a signal that induces transcription of the lacZYA operon. This initiates production of the proteins that
(A)
(B)
promoter architecture number of activators
(0, 0) activator
100
10-1
10-2
probability
promoter architecture
(0,0) (1,0) (0,1) (0,2) (2,0) (1,1) (3,0) (1,2) (0,3) (2,1) repressor
(1, 0)
(0, 1)
(1, 1)
number of repressors
Figure 1.6: Distribution of regulatory architectures in E. coli. (A) We classify regulatory architectures according to the number of activator sites Aand repressor sitesRin a promoter region, using the notation(A,R). This classification does not specify the positions of the binding sites. (B) We plot the frequencies of different regulatory architectures as noted in RegulonDB. Note that many promoters lack complete regulatory annotations, which skews the data towards (0,0).
enable the cell to metabolize lactose. In laboratory settings IPTG (isopropyl β-D-1- thiogalactopyranoside) is frequently used as an inducer for thelacZYAoperon instead of allolactose. IPTG is used because unlike allolactose, IPTG is not degraded by β-galactosidase, meaning that the concentration remains constant for the duration of the experiment.
ALLOSTERY LOOPING ARCHITECTURAL PROTEINS
SIMPLE ACTIVATION SIMPLE REPRESSION LacI
IPTG
CRP RNAP HU
Figure 1.7: Architecture of thelacZYAoperon. ThelacZYAoperon is regulated by CRP, which acts as a simple activator, and LacI, which acts as a repressor. LacI is an allosteric repressor that can adopt either an “active” conformation (red) that binds strongly to the DNA and prevents RNAP binding, or an “inactive” conformation (purple) that binds weakly to the DNA. When the ligand allolactose (or, alternatively, IPTG) binds to LacI, it stabilizes the inactive conformation and prevents repression.
LacI can perform either simple repression or repression by looping. Looping is facilitated by binding of the architectural protein HU.
Looping
Looping is a form of action at a distance in which a transcription factor binds simultaneously to two binding sites that are separated by hundreds of base pairs or more, which requires the intervening DNA to form a loop. Action at a distance is a common strategy employed by eukaryotic enhancers [29]. While only a handful of looping architectures have been studied in prokaryotes, a scan of RegulonDB indicates at least ∼ 50 instances of binding sites for the same transcription factor that are spaced approximately 90 bp apart, which is the minimum distance observed in well-studied natural looping architectures [30]. The prevalence of looping inE.
coli may be much higher than this, as Ref. [30] explored a narrow range of loop sizes and there are likely to be many transcription factor binding sites in theE. coli genome that are not currently reported in RegulonDB.
ThelacZYAoperon is a classic example of looping as a component of a regulatory architecture which has been studied extensively to better understand the physics of DNA bending in the context of gene regulation [7, 8, 31–39]. Looping in lacZYApromotes repression by increasing the local concentration of repressor [29]
and providing an additional state in which RNAP is prevented from binding to the promoter. Repression appears to be the most common usage of looping architectures in prokaryotes, though there are examples of looping being used for activation ofσ54- dependent transcription in a manner similar to eukaryotic enhancers, as reviewed extensively in Ref. [40].
Architectural Proteins
Bacteria possess a class of proteins that are analogous to histones in eukaryotes.
These proteins are alternatively known as architectural proteins, nucleoid-associated proteins, or histone-like proteins. Like histones, they are known to play a role in gene regulation (reviewed in Ref. [41]) and chromatin organization (reviewed in Ref. [42]). Some bind to specific DNA sequences, while others appear to bind nonspecifically or bind preferentially to bent DNA.
The architectural protein HU binds to deformed DNA and bends it. In thelacZYA operon, HU contributes to repression by binding to the looping region and facilitating looping between two LacI binding sites. Cells lacking HU exhibit significantly lower repression levels at thelacZYAoperon than cells possessing HU. Furthermore, while looping mechanics are known to depend on the DNA sequence of the looping region, bending due to HU does not appear to be affected by the relative “stiffness” of the DNA [8].
We discuss these examples to give a sense of the diverse modes of transcriptional regulation in prokaryotes, and we note that this is not a comprehensive list of types of transcriptional regulation. There are a number of other schemes that are worthy of discussion and have been addressed thoroughly elsewhere, including (but not limited to) regulatory role switching as in thearaBADoperon [27], toxin-antitoxin systems (see Chapter 3 for the example of therelBEoperon), the role of DNA shape [43], and altering DNA specificity using methylation [44].