Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Gene Expression
Regulation of Gene Expression at the Transcriptional Level

Every Cell of a whole Organism or population contains a complete set of genes characteristic of a given species (strain). However, at any given time, not all genes function (are expressed) in a cell, but only those whose products are needed. Such a distribution of "duties" among genes is possible due to the existence of Gene Expression regulation mechanisms that operate at different levels. Through these mechanisms, The Cell conserves its resources: at any specific moment, it synthesizes a defined, limited set of substances rather than their entire possible spectrum, and furthermore, coordinates metabolic pathways.

Among several levels of gene expression regulation, the Introduction/15.html">Regulation of enzyme synthesis, which is carried out at the METABOLISM/31.html">Transcription level, is the most essential and frequently utilized. The Essence of this type of regulation boils down to accelerating or slowing down the transcription processes of specific genes, which ultimately affects The rate of synthesis of their products. Positive and negative transcription regulation are distinguished. Negative regulation involves the inhibition of Transcription initiation through the binding of repressor Proteins to the operator region; positive regulation, conversely, encompasses events of transcription "activation" that are also caused by the attachment of specific proteins to the operator (in this case, called activators).

ENZYME SYNTHESIS REGULATION has been best studied in prokaryotes. Their characteristic feature is the Organization of genes involved in a single metabolic pathway into operons. This enables prokaryotes to "turn on" and "turn off" the transcription of a group of genes (belonging to the Operon) simultaneously. Several Different types of transcription initiation regulation (operon regulation) will be discussed in this topic using two Examples of E. coli operons: the lactose operon, which belongs to the group of catabolic operons, and the Tryptophan operon, which is an anabolic one.

The Regulation of transcription initiation in eukaryotes is carried out much more complexly than in prokaryotes, but its basic regularities are observed in this case as well.

Regulation of lactose operon expression by induction. The E. coli lactose operon contains a regulatory region (promoter and operator) and three structural genes: lacZ (encoding The Structure of ß-galactosidase), lacY (determining the structure of ß-galactoside permease), and lacA (the structure of ß-galactoside transacetylase) (Fig. 3.5). These Enzymes facilitate The transport of the disaccharide lactose into the cell and its Cleavage into glucose and galactose. Transcription of the structural GENES OF THE lactose operon is carried out in a coordinated manner: the lacZ, lacY, and lacA Genes are transcribed into a single polycistronic mRNA, which is translated to yield approximately equal amounts of each enzyme protein. Not far from the lactose operon on the E. coli chromosome is the I gene, which encodes the STRUCTURE OF THE repressor protein. In its free state, this protein has an affinity for the operator region of the lac operon.

When Escherichia coli Cells are grown on a medium with glucose as the sole carbon source, they contain very few proteins—the products of the structural genes of the lactose operon: approximately 10 molecules per cell. In the presence of lactose and other ß-galactosides, the concentration of these proteins increases to 10,000 or more molecules per cell. In this case, lactose (ß-galactosides) serves as an inducer of the synthesis of these enzymes, which means that the corresponding operon is regulated by induction, i.e., the enzymes it encodes are synthesized only in the presence of the inducer.

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Fig. 3.5. E. coli lactose operon (lac) and the closely linked lac repressor gene (lac I): P — promoter; O — operator

The induction mechanism is as follows. In the absence of the inducer, the free repressor protein (a tetrameric molecule) tightly binds to the operator region of the lac operon (Fig. 3.6). Since the promoter and operator sequences overlap, the binding of the repressor to the operator obstructs the attachment of RNA polymerase to the promoter, resulting in the blocking of structural gene transcription. However, the presence of lactose or another inducer of the lac operon in the cell leads to The formation of an inducer-repressor complex, which loses its affinity for the operator and frees the regulatory region (Fig. 3.6). Transcription of the structural genes takes place, and the corresponding proteins are synthesized on the formed mRNA.

Thus, the existence of the induction phenomenon allows the cell to conserve its resources, as it ensures that the transcription of inducible genes and the synthesis of corresponding enzymes do not occur constantly, but only when the inducer is present in the medium.

Mutations have been obtained in E. coli Bacteria that result in a decrease or disappearance of the repressor's affinity for the operator. In such mutants, a constitutive synthesis of lactose operon enzymes is observed, i.e., its expression is observed even in the absence of the inducer.

Regulation of enzyme synthesis by induction belongs to negative control. In addition, the operation of the lactose operon and many other operons is subject to positive regulation, which can be examined using catabolite repression as an example.

Fig. 3.6. Repression of the lac operon by the homotetrameric lac repressor (top) and Induction of the lac operon after binding of the ß-galactoside inducer to the repressor (bottom)

Regulation of lactose operon operation by catabolite repression. Positive regulation of lactose operon transcription consists in the binding of an activator complex to a specific sequence located at the very beginning of the lac promoter. This leads to The stimulation of lac operon transcription, As a result of which the rate of Synthesis of the corresponding mRNA increases almost 50-fold. This phenomenon does not yet have a definitive explanation, but there are several hypotheses describing the transcription stimulation process. According to the most widely accepted one, the activator complex binds to the part of the promoter that immediately adjoins the RNA polymerase attachment site (Fig. 3.7) and enhances the affinity of this enzyme for the promoter. An alternative hypothesis is that the binding of the activator complex to the promoter prevents RNA polymerase from attaching to a nearby weak promoter, thereby increasing the probability of the enzyme binding to the "correct" promoter site.

In this case, the function of the transcription activator is performed by a complex of cyclic AMP (cAMP) with the catabolite activator protein (CAP). This complex performs similar Functions in regulating the expression of many other catabolite operons. The free CAP protein is incapable of binding to the specific sequence within the promoter and requires the participation of cAMP.

cAMP is formed from ATP (Fig. 3.8) during enzymatic conversion in response to various cellular events and signals. This messenger molecule participates in many processes, through which The regulation of various facets of Cellular metabolism is carried out. The cAMP content in the cell is controlled by two balancing processes: synthesis involving adenylate cyclase (Fig. 3.8) and degradation by phosphodiesterase. Glucose accelerates cAMP breakdown and inhibits its synthesis, i.e., a low cAMP level is observed in the cell in the presence of glucose, and a high level in its absence.

Thus, the content of cAMP and, consequently, of the cAMP-CAP activator complex depends on the presence of glucose in the cell. In bacteria growing on glucose, the concentration of these substances is very low, therefore, even in the presence of Inducers, transcription of the lactose and similar operons does not occur, and enzymes participating in the Catabolism of corresponding sugars (lactose, arabinose, galactose, etc.) are not synthesized in the cell. This phenomenon is designated by the term "catabolite repression".

Regulation of tryptophan operon operation by repression. The E. coli tryptophan operon contains five structural genes (trpE, trpD, trpC, trpB, trpA) that determine The amino acid

Fig. 3.7. Nucleotide sequences participating in the regulation of lactose operon expression

Fig. 3.8 Synthesis of cyclic AMP (cAMP) from ATP

sequences of five enzymes involved in The conversion of chorismate to tryptophan. In addition, the operon includes the trpL leader segment and a regulatory region (overlapping promoter and operator sequences) that participate in the regulation of structural gene transcription (Fig. 3.9). At another site of the E. coli nucleoid (at a sufficient distance from the trp operon) is located the trpR gene, which encodes the structure of the repressor protein.

The structural genes of the trp operon are transcribed as a polycistronic mRNA 7,000 NUCLEOTIDES long. mRNA synthesis is initiated at a promoter whose sequence overlaps with the operator (Fig. 3.9). Transcription is controlled by the interaction of the repressor protein with the operator, whose effector is the end product of this biosynthetic pathway—tryptophan. When free tryptophan is present in the cell, it binds to the repressor and exerts an allosteric effect on the latter's structure, as a result of which the repressor acquires The ability to tightly bind to the operator. In this case, tryptophan acts as a corepressor of the tryptophan operon.

The binding of the tryptophan-repressor complex to the operator region prevents the proper interaction of RNA polymerase with the promoter, as the operator and promoter sequences overlap. As a result, transcription of the trp operon is blocked, and the enzymes involved in tryptophan synthesis are not produced.

In the absence of tryptophan, the free repressor protein does not bind to the operator, and operon transcription proceeds unhindered. Under these conditions, there is an intensive synthesis of the five enzymes that convert chorismate into tryptophan. Due to regulatory mechanisms, the concentration of these enzymes in an E. coli cell can vary up to 700-fold depending on the intracellular tryptophan level.

Fig. 3.9. The E. coli tryptophan operon (trp operon). Structural gene products: a1 — anthranilate synthase, component I; a2 — anthranilate synthase, component II — phosphoribosylanthranilate transferase; pr — phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthase; t1 — tryptophan synthase ß; t2 — tryptophan synthase a. Intermediates: PR — phosphoribosyl; CdRP — carboxyphenylamino deoxyribulose phosphate; InGP — indoleglycerol phosphate; PRPP — phosphoribosyl pyrophosphate

Repression also regulates The activity of other operons, particularly those involved in The Biosynthesis of Other Amino Acids. Some of these operons feature an additional regulatory mechanism known as attenuation.

Attenuation of tryptophan operon expression. This mechanism of regulating trp operon expression couples two processes: transcription and Translation. It involves a regulatory DNA segment located upstream of the structural gene trpE. This so-called trpL leader segment contains an attenuator sequence approximately ~ 145 nucleotide pairs in length. When free tryptophan is present in the cell, transcription of the attenuator sequence proceeds, followed by its premature termination, releasing the ~145-nucleotide trp leader mRNA. In the absence of tryptophan, premature termination does not occur, and full-length tryptophan mRNA is transcribed. Thus, the attenuator sequence contains a signal that regulates the transcription of structural genes.

Sequence Analysis of the attenuator sequence revealed three unusual segments: one encodes a short polypeptide consisting of 14 amino acids, including two adjacent tryptophan residues; the other two segments contain inverted repeats capable of forming hairpin structures of varying configurations through complementary nucleotide pairing. One of these two hairpin structures does not interfere with transcription, whereas the second mediates p-independent premature transcription termination. Which of the two hairpin structures forms depends on the intracellular tryptophan concentration: in its absence (or at low concentrations), the short peptide containing the two tandem repeats of this amino acid cannot be fully synthesized, which serves as a signal for the Formation of the non-terminating hairpin that allows transcription to continue. When cellular tryptophan levels are moderate to high, the short peptide encoded by the attenuator sequence is synthesized; in this case, Ribosomes reach the stop codon in the leader mRNA, triggering the formation of the hairpin structure that halts further transcription. Thus, attenuation of premature transcription termination occurs when tryptophan is low or nearly absent in the cell, signaled by the specific position of the ribosome on the leader mRNA.

Expression of the tryptophan operon, as well as several other anabolic operons (Histidine, Threonine, isoleucine-valine), reaches its maximum level in the absence of repression and with maximal attenuation of transcription termination.