Fundamentals of Molecular Biology - V.I. Rezyapkin 2009

Regulation of Gene Expression
Regulation of Gene Expression at the Transcriptional Level

Prokaryotes

METABOLISM/31.html">Transcription in prokaryotes can be regulated at the initiation, elongation, and termination stages. Most frequently, transcription is regulated at the Transcription initiation stage. Regulation at the transcription initiation level

The efficiency of transcription initiation depends largely on promoter strength. The stronger the promoter, the more efficient RNA Synthesis is. The Specificity of prokaryotic RNA polymerase binding to the promoter is determined by the σ-subunit. Bacterial Cells contain several different σ-subunit variants that drive transcription from various genes. The σ-subunit is required solely for promoter recognition and does not participate in RNA synthesis itself. Promoters recognized by the same σ-subunit may differ slightly from one another in their Primary Structure. These variations determine promoter strength. Interestingly, the closer a native promoter's structure is to the consensus promoter, the stronger that promoter tends to be.

Prokaryotic Cells possess Proteins that regulate transcription efficiency: repressor proteins, which block RNA synthesis, and activator proteins, which positively influence RNA synthesis.

In prokaryotes, transcription and Translation are coupled processes: RNA synthesis is still underway when Ribosomes already begin synthesizing the polypeptide (Fig. 8.4). Interestingly, successful translation actually promotes transcription. The ribosome effectively pushes the RNA polymerase forward. In the absence of translation, RNA synthesis can be blocked, whereby a specialized protein "pulls off" the nascent RNA.

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Fig. 8.4. In prokaryotes, transcription and translation are coupled processes

The activity of many promoters is regulated by specialized regulatory proteins: Repressors and activators. Repressors interact with DNA to reduce RNA polymerase activity, which is referred to as negative Transcriptional Regulation. The DNA region that binds the repressor is called the operator. There are also low-molecular-weight substances affecting the repressor's affinity for the operator, known as effectors. Effectors are divided into two types: Inducers and corepressors. Inducers decrease the affinity of the repressor for the operator, whereas corepressors increase it. Activators, by contrast, enhance RNA polymerase activity, thereby exerting positive Introduction/30.html">Regulation of Gene Expression. Some proteins can act as repressors in certain contexts and as activators in others.

Next, we will examine the Regulation of the lactose and Tryptophan operons in *E. coli*.

The Lactose Operon (*lac* operon) of *E. coli*

This operon encodes proteins involved in lactose Catabolism—lactose being an energy source less preferred by this bacterium than glucose. Consequently, Transcription of the *lac* operon occurs only when glucose is absent and lactose is present in the medium. The *lac* operon includes (Fig. 8.5): three structural genes encoding proteins responsible for lactose metabolism—ß-galactosidase (which hydrolyzes lactose), permease (responsible for transporting galactose into The Cell), and protein A (ß-galactoside transacetylase, whose exact function remains to be fully elucidated); a promoter (40 bp); an operator (27 bp); and the binding site for the cAMP-CRP complex (catabolite activator protein)—CRP*cAMP (38 bp).

Fig. 8.5. Organization OF THE *lac* operon

Transcription of the *lac* operon depends on the presence or absence of lactose and glucose in the growth medium of *E. coli* cells. Let us consider three scenarios.

1) Both lactose and glucose are absent from the growth medium of *E. coli* cells.

Transcription of the repressor gene yields an mRNA whose translation produces the repressor protein. The latter interacts with the operator, preventing

RNA polymerase from initiating RNA synthesis (Fig. 8.6). Under these conditions, the proteins responsible for lactose utilization are not synthesized. This makes perfect sense: if lactose is absent, synthesizing proteins to metabolize it would be pointless.

Fig. 8.6. In the absence of lactose in the medium, the repressor interacts with the operator and prevents RNA polymerase from initiating RNA synthesis

2) Lactose is present and glucose is absent in the growth medium of *E. coli* cells. Upon entering the bacterial cell, lactose binds to the repressor (Fig. 8.7A). The resulting repressor-lactose complex loses its affinity for the operator and dissociates from it (Fig. 8.7B). When glucose is scarce, cAMP begins to accumulate within the cell; by binding to the CRP protein, it forms the CRP*cAMP complex (Fig. 8.7C). This complex interacts with the corresponding DNA region, facilitating the initiation of lactose operon transcription by RNA polymerase (Fig. 8.7D). As a result, mRNA is synthesized and used as a template for The production of proteins (Fig. 8.7E) responsible for lactose catabolism. The cell is now equipped to use lactose as an energy source. It should be emphasized that *lac* operon transcription is highly efficient specifically when glucose is absent and lactose is present in the medium.

Fig. 8.7. Transcription of the *lac* operon occurs only in the absence of glucose and the presence of lactose

3) Both lactose and glucose are present in the growth medium of *E. coli* cells

In this scenario, the operator is not bound by the repressor because the latter is complexed with lactose and lacks affinity for the operator. At the same time, RNA polymerase cannot efficiently carry out RNA synthesis because intracellular cAMP concentrations are insufficient to form the CRP*cAMP complex required for efficient transcription. Thus, in the presence of both glucose and lactose, the *lac* operon is virtually untranscribed, and lactose catabolic proteins are not synthesized. Under these circumstances, the cell preferentially utilizes glucose rather than lactose as its energy source. The Tryptophan Operon (*trp* operon) of *E. coli*

The tryptophan operon contains 5 genes responsible for tryptophan Biosynthesis, a promoter, and an operator (Fig. 8.8).

Fig. 8.8. Schematic organization of the E. coli tryptophan operon

Obviously, the tryptophan operon should be transcribed when tryptophan levels in the cell are low. Conversely, when the concentration of this amino acid is high, its expression should be minimal or completely absent. E. coli cells contain a repressor produced through the expression of the corresponding gene. When intracellular tryptophan is abundant, this repressor forms a complex with The amino acid. The resulting complex exhibits high affinity for the operator and, by binding to it, prevents RNA polymerase from initiating RNA synthesis (Fig. 8.9). The proteins responsible for tryptophan synthesis are not produced, and consequently, the Synthesis of the amino acid is halted.

Fig. 8.9. When intracellular tryptophan levels are high, the tryptophan operon is not transcribed

As soon as the tryptophan concentration in the cell drops, the tryptophan-repressor complex dissociates. As a result, the repressor loses its affinity for the operator. RNA polymerase gains access to the promoter and initiates transcription of the structural genes. mRNA is synthesized and subsequently translated to produce the Enzymes responsible for tryptophan biosynthesis. The concentration of this amino acid in the cell gradually increases (Fig. 8.10).

Fig. 8.10. In the absence of tryptophan, the trp operon is expressed

Transcriptional Regulation at the Elongation Level: Attenuation

We have previously discussed The regulation of the tryptophan operon via the repressor protein. However, the regulation of this operon can also be mediated by an attenuator sequence (attenuator) located within it. This sequence is part of the leader region, which also encodes a leader peptide (Fig. 8.11). The leader peptide contains two tryptophan residues at positions 10 and 11.

Fig. 8.11. STRUCTURE OF THE leader region of the tryptophan operon

Transcription of the trp operon may or may not be prematurely terminated at the attenuator region. Premature termination is observed when intracellular tryptophan levels are high, since an Abundance of this amino acid eliminates The Need for its further synthesis and, consequently, for the enzymes that produce it. Conversely, at low tryptophan concentrations, transcription of the operon proceeds to completion (Fig. 8.12). This results in the synthesis of mRNA, which is translated into enzymes required for The biosynthesis of this amino acid.

Fig. 8.12. At high tryptophan concentrations, trp operon transcription is prematurely terminated at the attenuator, whereas in the absence of tryptophan, it proceeds fully

Translation of the mRNA leader region is coupled with transcription. The rate of its translation depends on the tryptophan content in the cell. At high concentrations of this amino acid, the ribosome rapidly translates this region of the mRNA.

A high translation rate promotes The formation of structures that lead to premature termination of transcription. Conversely, if the ribosome translates the leader mRNA region slowly (at low tryptophan concentrations), a structure is formed that facilitates the continuation of transcription. As a result, mRNA is synthesized and translated into the proteins responsible for tryptophan biosynthesis. Regulation at the Termination Level

Typically, RNA synthesis terminates at the terminator region. However, in some cases, RNA polymerase bypasses the terminator and reads through it, continuing RNA synthesis (Fig. 8.13) beyond its boundaries. This process is known as antitermination. Antitermination is mediated by specialized antiterminator proteins, allowing the transcription of genes located downstream of the terminator.

Fig. 8.13. Antitermination. A - RNA synthesis terminates at the terminator region. B - RNA polymerase, assisted by antiterminator proteins, transcribes genes located downstream of the terminator. P - promoter, T - terminator.

Eukaryotes

The efficiency of Gene Expression at the transcriptional level in eukaryotes depends on various factors. The roles of promoters, enhancers, silencers, and transcription factors were discussed earlier in the section "Transcription." In this section, we will examine other mechanisms of gene expression regulation in greater detail.

Effect of Steroid Hormones on Gene Expression

Steroid hormones regulate numerous biological processes in eukaryotic organisms. Their action is mediated through their effects on the cellular genetic apparatus.

Steroid hormones are hydrophobic and can therefore easily cross cell membranes into the Cytoplasm. Here, the hormone binds to a receptor protein, which essentially Functions as a transcription factor, forming a hormone-receptor complex. This complex enters the Cell Nucleus, where it interacts with specific nucleotide sequences to activate the transcription of steroid hormone-dependent genes. This activation leads to the synthesis of mRNA, which is transported to the cytoplasm, where it is translated into proteins (Fig. 8.14). Thus, steroid hormones direct the synthesis of proteins that ultimately determine their biological effects. It should be noted that in some cases, the hormone-receptor complex represses the expression of certain genes.

Fig. 8.14. MECHANISM OF ACTION of steroid hormones

Effect of THYROID HORMONES on gene expression

The thyroid hormones thyroxine and triiodothyronine regulate numerous biological processes in Human and Animal organisms. These hormones are derivatives of the amino acid Tyrosine (Fig. 8.15).

Fig. 8.15. Thyroid hormones.

Figure 8.16 illustrates The Mechanism of action of thyroxine. Like steroid hormones, thyroxine acts at the genetic level. Upon penetrating The cell membrane, it undergoes deiodination and is converted into triiodothyronine. The latter forms a complex with its receptor and activates the transcription of specific genes. The synthesized mRNA is transported from The Nucleus to the cytoplasm, where it serves as a template for Protein Synthesis. The newly formed proteins mediate the hormonal effects of thyroxine.

Fig. 8.16. Mechanism of thyroxine action. T₄ - thyroxine, T₃ - triiodothyronine.

Effect of hormones whose receptors are located on the cell membrane on gene expression

Only a small fraction of hormones—specifically, steroid and thyroid hormones—penetrate the cell nucleus. The majority of hormones regulate gene expression without entering the cell, exerting their effects on the genetic apparatus indirectly through receptors located on The Plasma Membrane. Several pathways of signal Transduction from Membrane Receptors to the nucleus are known.

In the first pathway (Fig. 8.17), the interaction of the hormone with its receptor activates protein kinase, an enzyme that catalyzes protein phosphorylation (the attachment of a phosphate group to amino acid residues in the protein molecule). Protein Kinases utilize ATP as a phosphate group donor:

The activated protein kinase enters the nucleus, where it phosphorylates one or more intranuclear transcription factors. Phosphorylation alters the affinity of these factors for DNA and modifies their activity, leading to the activation of specific genes and the synthesis of mRNA. The mRNA is then transported to the cytoplasm, where it acts as a template for the Synthesis of Other transcription factors. These factors subsequently translocate into the cell nucleus and activate late-response genes. The products of these genes determine the BIOLOGICAL EFFECTS OF the hormones (Fig. 8.17).

Fig. 8.17. Signal transduction from the hormone to the cell's genetic apparatus via the receptor, mediated by protein kinase through the phosphorylation of an intranuclear transcription factor, after entering the nucleus. PK - protein kinase, TF - transcription factor, P - phosphate group.

In the second pathway (Fig. 8.18), the hormone indirectly activates protein kinase via the receptor, which in turn phosphorylates a cytoplasmic transcription factor. Following phosphorylation, this factor translocates into the cell nucleus, binds to DNA, and triggers a series of events similar to those described above.

Fig. 8.18. Signal transduction from the hormone to the cell's genetic apparatus via the receptor, mediated by a protein kinase-phosphorylated transcription factor. PK - protein kinase, TF - transcription factor, P - phosphate group.

In the third pathway, following the hormone-receptor interaction, protein kinase phosphorylates an inhibitory subunit within the inhibitor-transcription factor protein complex (Fig. 8.19). This phosphorylation causes the complex to dissociate. The released transcription factor enters the nucleus and, upon binding to DNA, initiates a cascade of events that result in the cellular biological response to the hormone.

Fig. 8.19. Upon receiving the hormonal signal, protein kinase phosphorylates the inhibitory subunit of the inhibitor-transcription factor complex. This phosphorylation leads to the dissociation of the complex. The released transcription factor translocates into the nucleus and binds to DNA, triggering a sequence of events that elicit the cellular biological response to the hormone. PK - protein kinase, TF - transcription factor, P - phosphate group, I - inhibitor, I-TF - inhibitor-transcription factor complex.

Across all three pathways of hormonal signal transduction to the cellular genetic apparatus, primary transcription factors bind to DNA and initiate the transcription of early-response genes. The products of these genes are secondary transcription factors that stimulate the expression of late-response genes, which ultimately determine the cell's biological response to the hormone.