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E. coli contains ~ 4,000 genes and ~ 107 protein molecules per cell. If each gene is expressed at the same level, then each protein would be present in 107/4,000 = 2,500 copies. However, this is not observed. Instead, some genes are expressed more than others. For instance, there are more than 10,000 copies of ribosomal proteins but only 10 copies of lac repressor per cell. Clearly, cells are capable of differentially regulating gene expression. In addition, cells can respond to environmental cues by altering their pattern of gene expression.
The predominant sigma factor used by E. coli is sigma 70. Under special conditions, an alternative sigma factor can be made. Alternative sigma factors recognize unique promoter sequences and so direct RNA polymerase to transcribe a different set of genes than sigma 70. This system allows the coordinate, global regulation of many genes required under a set of specific conditions, e.g. sigma 54 is used under conditions of nitrogen starvation.
Cells constantly make decisions about which proteins to make, and they are very economical.
| Positive Regulation: | Negative Regulation: | |
| 1. Purpose | Primarily for decisions about utilizing the best sources of carbon, nitrogen, electron donor, electron acceptor, etc. | Primarily for decisions about whether to synthesize something that might be available from the environment. |
| 2. Protein required | An activator | A repressor |
| 3. Effectors: small molecules that act as signals. | Inducer - In positive regulation, an inducer will bind and activate the activator protein. | a. Co-repressors - repressors may not function unless they are
first bound to a small co-repressor molecule.
b. Inducer - repression may be relieved when a small inducer binds and inactivates the repressor. |
| 4. Relevant DNA sequences - | Specific activator binding sites are recognized by activator proteins. | Specific operator sites are recognized by repressors. |
| 5. Examples: | a. The activator protein CAP (Catabolite Activator Protein)
of E. coli interacts with the inducer cAMP. The protein subsequently
binds CAP binding sites in DNA and stimulates transcription of downstream
genes. This system is involved in the use of alternate carbon sources when
the primary source, glucose, is in short supply.
b. Rather than being activated by the binding of an inducer, the activator protein virG of Agrobacterium tumefaciens is activated by phosphorylation in response to substances released from wounded plants. It subsequently directs the transcription of other genes involved in the infection of the plant. |
a. The trp repressor binds the co-repressor
tryptophan and represses the transcription of genes encoding enzymes involved
in tryptophan biosynthesis.
b. The lac repressor binds the inducer (allo)lactose. This relieves repression of transcription of the lac operon and allows the expression of genes encoding proteins involved in the metabolism of lactose when other carbon sources (e.g. glucose) are not available. |
Operon - a group of genes jointly transcribed and whose expression
is jointly regulated. This is a prokaryotic feature.
The lac operon contains genes encoding proteins required to metabolize
the sugar lactose.
1. Glucose is the favorite carbon source of E. coli. However, other sugars such as lactose can be utilized if glucose is in short supply.
2. Relevant molecules (see handout):
a. Lactose - a disaccharide containing one glucose and one galactose residue. In order for it to serve as a carbon/energy source, the enzyme beta-galactosidase must cleave lactose into its component monosaccharides and the galactose must be isomerized to form glucose.
b. cAMP - a "starvation signal"; elevated levels of this molecule indicate that the supply of glucose is low.
c. ONPG (ortho-nitrophenyl-beta-D-galactopyranoside) - an artificial substrate for beta-galactosidase; when cleaved, a yellow product is released that can be detected spectrophotometrically. This provides a convenient way to measure the activity of the enzyme.
d. IPTG (isopropyl-beta-D-thiogalactopyranoside) - another artificial substrate; although IPTG cannot be cleaved by beta-galactosidase, it can act as an inducer. It is therefore known as a "gratuitous inducer".
3. Some observations: E. coli growth curves in the presence of various carbon sources (Figure 5.48)
In cultures containing glucose and lactose, the glucose is completely exhausted before the cells begin utilizing the lactose. There is a pause in growth during the transition from metabolizing the two carbon sources, during which time the cells are synthesizing the proteins required for using lactose (i.e. the lac operon is being expressed). When beta-galactosidase activity is measured, for example with ONPG, activity is only detected during the time that lactose is being metabolized - no activity is seen before this.
4. Catabolite repression and cAMP/CAP activation
When bound with cAMP, CAP binds the CAP binding site located just ahead of the promoter (Figure 5.44). This facilitates the binding of RNA polymerase at the promoter. It is important to note that this positive regulatory system is necessary but not sufficient to express the lac operon. Utilizing carbon sources other than glucose requires two signals:
5. Induction of the lac operon: relief of repression
The mal operon contains genes encoding proteins required to metabolize the sugar maltose.
1. cAMP/CAP activation
The mal operon is under the same sort of positive regulation as the lac operon - a CAP binding site is found just ahead of the mal promoter. Thus, in the presence of the cAMP "starvation signal", cAMP/CAP binds upstream of the mal operon as well as the lac operon (and many others too). As noted above, this "first signal" is necessary but not sufficient to activate the operon. The cell must "choose" whether to express the mal operon, the lac operon or one of the others. The choice, of course, will depend on what sugar is available to consume - maltose, lactose or something else.
2. Induction of the mal operon: activation by an activator protein
The mal and lac operons differ in the method used by the inducer sugar (the "second signal") to express the operon. Whereas the lac operon uses the inducer (allo)lactose to relieve repression by removal of the lac repressor, the mal operon uses the inducer maltose to activate an activator protein which binds an activator binding site and thus induces expression of the mal operon. Activator proteins induce expression by assisting RNA polymerase in promoter recognition. In addition, activator binding sites may be located far away from the genes they activate. In these cases, the DNA "loops" around so that the activator may contact the RNA polymerase (Figure 5.45).
This system uses a pair of modular proteins to detect and respond to specific environmental signals. The pair of proteins are a sensor kinase and a response regulator. Each of these proteins is in turn composed of two regions. The sensor kinase contains one domain that senses the signal and a second domain that has kinase activity (a kinase is an enzyme that phosphorylates targets). Upon sensing a specific signal, the sensor kinase autophosphorylates itself at a conserved histidine using the gamma phosphate of ATP. The phosphate is then transferred to a conserved aspartate residue located in the first domain of the response regulator. The second domain of the response regulator is responsible for bringing about the cell's response to the signal. Often, phosphorylation of the response regulator activates latent transcriptional regulatory activities within the protein. Thus, the response to the environmental signal often involves altering the pattern of gene expression.
Examples: