Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Control of Gene Expression
Regulation of Prokaryotic Transcription

The synthesis of mRNA and, consequently, Protein Synthesis must be tightly regulated, since a Cell lacks the resources required for the simultaneous METABOLISM/31.html">Transcription and Translation of all structural genes.

Both PROKARYOTES AND EUKARYOTES continually synthesize only those mRNAs that are essential for carrying out basic cellular Functions.

The expression of remaining structural genes is under strict control by regulatory systems that initiate transcription only when There is a demand for a specific protein.

The very "decision" to initiate transcription of a particular Gene forms the foundation of the entire subsequent mechanism of protein synthesis. Through transcriptional initiation control, The Cell can fine-tune its protein composition.

Gene transcription repression results in low-level gene transcription, whereby the corresponding mRNA is synthesized at a slow rate.

Gene transcription activation leads to robust mRNA transcription and, correspondingly, to the intensive Synthesis of the target protein.

In most Bacteria and other unicellular organisms, The rate of Gene Expression is efficiently regulated to allow fine-tuning of enzymatic systems and structural components, thereby enabling an adequate response to environmental fluctuations. At any given moment, a bacterial cell synthesizes precisely the Proteins that ensure its survival under those specific conditions.

In Multicellular Organisms, the Control of Gene Expression is primarily geared toward ensuring that the appropriate gene is activated in the correct cell at the right time during embryonic development and tissue differentiation.

In prokaryotes, Transcription is initiated by the binding of RNA polymerase to the TATA and TTGAC sequences within the pre-promoter region of a structural gene or Operon.

The promoters of most E. coli structural genes contain two binding sites for RNA polymerase. One of these (the TATA box, or Pribnow box) typically consists of The nucleotide sequence

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and the other is

The TATA box and the TTGAC sequence are located approximately 10 NUCLEOTIDES (the -10 region) and 35 nucleotides (the -35 region) upstream of the Transcription initiation site (nucleotide +1), respectively (Figure 19).

Figure 19 - Schematic diagram of a prokaryotic operon

Typically, the region (o) between the TATA box and the +1 nucleotide largely determines whether transcription of a given operon will take place.

Depending on the mode of Transcriptional Regulation of the operon, this region is referred to as an operator or an activator.

We will examine The Mechanism of transcriptional control in bacteria using the E. coli lac operon as an example.

In E. coli, approximately half of all genes are organized into operons that encode Enzymes involved in specific metabolic pathways or assemble post-translationally into a single multisubunit protein.

For example, the trp operon discussed above encodes five enzymes required for Tryptophan synthesis.

Similarly, the lac operon contains three genes: lac z, lac y, and lac a, which encode three enzymes: ß-galactosidase, ß-galactoside permease, and transacetylase, involved in the metabolism of lactose (a sugar present in milk, Figure 20). Because a bacterial operon is transcribed as a single contiguous unit from a single start point into one mRNA molecule, the expression of all genes within the operon is coordinately regulated—they are either all repressed or all activated together.

Figure 20 - Cytology/cytology/92.html">SCHEMATIC Structure OF the disaccharide lactose, composed of galactose (left) and glucose (right)

Transcription of operons (or isolated genes not included in operons) is controlled by the interaction between RNA polymerase and specific repressor or activator proteins.

In addition, to initiate transcription, E. coli RNA polymerase must associate with one of several small proteins known as σ factors, most commonly the σ⁷⁰ factor.

As long as E. coli is in a lactose-depleted medium, the synthesis of lac mRNA is repressed, preventing the cell from wasting resources on enzymes that are not currently needed.

In a medium containing both lactose and glucose, E. coli preferentially metabolizes glucose. Full-scale lactose metabolism is only activated when lactose is abundant and glucose is nearly depleted. This Metabolic Regulation is achieved by repressing lac operon expression until lactose becomes available, while keeping lac mRNA synthesis at a low baseline level until the glucose concentration drops (Figure 21).

Figure 21 - Regulation of E. coli lac operon expression

Transcription of the lac operon is controlled by the lac repressor and the catabolite activator protein (CAP), each of which binds to specific nucleotide sequences within the regulatory region of the lac operon (Figure 21(a)).

For transcription of the lac operon to begin, the σ⁷⁰ subunit of RNA polymerase must bind to the lac promoter located immediately upstream of the transcription start site.

In the absence of lactose, the lac repressor binds to the lac operator, which is situated immediately downstream of the promoter, even slightly overlapping it.

The binding of the lac repressor to the lac operator blocks the promoter, preventing RNA polymerase from even binding to it (Figure 21(b)). Conversely, when lactose is present in the medium, it binds to specific sites on each of the four subunits of the lac repressor, inducing a conformational change that causes the repressor to dissociate from the lac operator. As a result, RNA polymerase can successfully initiate transcription of the lac operon (Figure 21(b)).

At the same time, as long as glucose is present in the medium, the rate of transcription initiation (i.e., the number of initiation events per unit time) remains extremely low. Consequently, only a small number of mRNA molecules and proteins encoded by the lac operon are synthesized.

As soon as glucose is removed from the medium—a process accompanied by a drop in intracellular glucose levels—E. coli synthesizes cyclic adenosine monophosphate, cAMP (Figure 22).

Figure 22 - Cyclic adenosine monophosphate, cAMP

As the cAMP concentration rises, it binds to each subunit of the dimeric CAP protein, inducing a conformational change that enables the protein to bind to the CAP site of DNA within the region governing transcription of the lac operon (Figure 21 (d)).

The CAP-cAMP complex interacts with RNA polymerase bound to the promoter, significantly boosting the rate of transcription initiation (by minimizing abortive initiation). This activation leads to robust transcription of mRNA and, consequently, to the intensive synthesis of the enzymes encoded by the lac operon.

Once the enzymes have metabolized all the lactose, the lactose-free repressor switches off the lac operon.

Although the promoter structures of various E. coli genes are similar (i.e., homologous), their exact nucleotide sequences differ. It is precisely the nucleotide sequence within the promoter that dictates the rate of transcription initiation in the absence of Repressors and activators.

Promoters that sustain a high rate of transcription initiation are referred to as strong promoters.

Promoters that sustain a low rate of transcription initiation are referred to as weak promoters. For instance, the lac operon is a classic example of a weak promoter; its inherently low transcription initiation rate is further reduced by the lac repressor or significantly enhanced by the CAP-cAMP activator.