BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 6. AMINO ACID METABOLISM AND FUNCTIONS. PROTEIN BIOSYNTHESIS

6.10. Protein biosynthesis

6.10.10. Regulation of protein synthesis

The primary prerequisite for the existence of living organisms is the coordinated functioning of interconnected elements within a complex regulatory system, particularly at the transcriptional and translational levels. This enables organisms to adapt to novel environments, allowing microorganisms to acclimate to their surrounding nutrient medium or complex Multicellular Organisms to meet new physiological demands under altered living conditions.

Cells of living organisms possess the capacity to synthesize a vast array of diverse Proteins, yet they never express all of them simultaneously. The Abundance and Diversity of proteins, particularly Enzymes, are determined by their degree of involvement in METABOLISM. Furthermore, metabolic intensity is regulated by The rate of Protein Synthesis while being concurrently controlled via allosteric pathways. Consequently, Protein synthesis is modulated by external and internal factors, as well as by conditions wherein The Cell produces the precise quantity and repertoire of proteins required to perform specific physiological Functions.

The general theory of the Regulation of Protein Synthesis was formulated by French scientists and 1965 Nobel Prize laureates in Physiology or Medicine, F. Jacob and J. Monod. The Essence of this theory lies in the "switching on" or "switching off" of genes as functional units, determining whether they can manifest their ability to transmit the Genetic information encoded in DNA regarding the Synthesis of specific proteins. This proposed mode of regulation, validated through experiments on Bacteria, was designated as Transcriptional Regulation of protein synthesis, because alterations in the Rate of protein synthesis occur through Changes in the rate of Gene Transcription, specifically at the stage of mRNA formation.

Studies conducted on E. coli cells have demonstrated that bacteria contain three distinct types of enzymes:

✵ constitutive enzymes - present in cells at a constant level regardless of the metabolic state of the Organism;

✵ inducible enzymes - their concentration is normally low but can increase significantly (up to 1,000-fold or more) upon The addition of enzyme substrates to the culture medium;

✵ repressible enzymes - metabolic pathway enzymes whose synthesis is halted when the end product of those pathways is added to the medium.

Thus, enzyme induction (de novo enzyme synthesis) has been proven in bacteria upon the Introduction of their substrates into the culture medium. Conversely, the addition of end products generated by Reactions Catalyzed by these very enzymes leads to a reduction in the amounts of synthesized enzymes. The latter phenomenon is termed enzyme repression. Both phenomena—Induction and Repression—are mutually interdependent.

According to the theory of F. Jacob and J. Monod, bacterial Protein Biosynthesis involves at least Three types of genes: structural genes, regulator genes, and operator genes. Structural genes determine the Primary Structure of the synthesized protein. Within the DNA strand, they serve as the template for mRNA biosynthesis, which subsequently enters the ribosome and acts as a template for protein synthesis. The regulation of protein synthesis via induction is illustrated in Fig. 6.47.

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Fig. 6.47. Regulation of protein synthesis via induction (schematic):

RG - regulator gene; P - promoter; OG - operator gene

The synthesis of mRNA on structural genes is directly controlled by a specific segment of the DNA molecule known as the operator gene. It acts as the trigger mechanism for the functioning of structural genes. The operator gene is located adjacent to the structural genes it regulates. The reading of The Genetic Code—namely, The formation of mRNA—begins at the promoter, a DNA region situated next to the operator gene that serves as the initiation point for mRNA synthesis, and then proceeds sequentially along the operator and structural genes. Therefore, the transcription of structural genes depends on the ability of RNA polymerase to bind to the promoter. The synthesized mRNA molecule encoding the synthesis of several different proteins is referred to as a polygenic (polycistronic) transcript. A single gene or a group of structural genes coordinated by a single operator (often genes encoding proteins whose functions are closely linked in metabolic processes) constitutes an Operon.

The operation of the operon is subject to the controlling influence of another region of the DNA strand—the regulator gene. Structural genes and the regulator gene are located in different Regions of the DNA chain; thus, as hypothesized by F. Jacob and J. Monod, the communication between them is mediated by an intermediary protein called a repressor. The Synthesis of the repressor occurs on a specific mRNA template transcribed from the regulator gene (Fig. 6.48). The repressor is synthesized at a constant rate within the cell and exhibits a high affinity for the operator gene, reversibly binding to it to form a complex. The formation of this complex leads to the blockade of mRNA synthesis and, consequently, of protein synthesis; thus, the function of the regulator gene is to arrest The activity of structural genes transcribing mRNA via the repressor protein. The structural regions of the promoter and operator may partially overlap, meaning that the binding of the repressor protein to the operator creates steric hindrance for RNA polymerase attachment.

Furthermore, the repressor possesses The ability to bind with strict Specificity to certain low-molecular-weight substances known as Inducers or effectors. When such an inducer binds to the repressor, the latter loses its ability to bind to the operator gene, which consequently escapes the control of the regulator gene, ultimately allowing mRNA synthesis to commence. This is a classic example of negative control, wherein the inducer, by binding to the repressor protein, induces Conformational Changes in its tertiary structure such that the repressor loses its capacity to bind to the operator gene.

Fig. 6.48. Regulation of protein synthesis via repression:

RG - regulator gene; P - promoter; OG - operator gene

The mechanisms of the described protein synthesis regulation and the interaction between the repressor and structural genes were proposed based on data derived from studying the lactose operon (lac operon) of E. coli, which encodes proteins involved in lactose utilization. One of the three proteins facilitating lactose utilization is β-galactosidase (lactase), an enzyme that cleaves milk sugar into glucose and galactose. Wild-type E. coli typically grows on glucose and contains about 10 molecules of these enzymes per cell. If lactose (a novel source of energy and carbon) is added to the medium instead of glucose, the strain will not grow until the corresponding enzymes are synthesized (adaptive synthesis). The amount of each of the three enzymes must increase to 5,000. Upon The entry of lactose (the inducer) into the cell, its molecules bind to the repressor protein and disrupt the linkage between the repressor and the operator gene. The operator gene and structural genes thereby resume functioning and synthesize the requisite mRNA, which "instructs" Ribosomes to synthesize β-galactosidase. Simultaneously, the regulator gene continues to stimulate repressor production, but the repressor is blocked by new lactose molecules, allowing the synthesis of the β-galactosidase enzyme to persist. As soon as the lactose molecules are completely hydrolyzed, the repressor is released and, reaching the DNA, binds the operator gene to block mRNA synthesis and, consequently, the ribosomal synthesis of β-galactosidase.

Thus, The biosynthesis of mRNA controlling Protein synthesis in ribosomes depends on the functional state of the repressor—a tetrameric protein with a total Molecular Weight of 1.5·10⁵. When it is in an active state, meaning unlinked to an inducer, it blocks the operator gene, and mRNA synthesis does not occur. Upon the arrival of an inducer in the cell, its molecules bind the repressor, converting the latter into an inactive form (or potentially reducing its affinity for the operator gene). The structural genes are thus released from repressive control and begin synthesizing the required mRNA.

As previously noted, the concentration of a series of enzymes in cells drops sharply when the levels of end products generated in a sequence of enzymatic reactions increase. This phenomenon, termed enzyme repression, is frequently observed in biosynthetic pathways. In such cases, repressor molecules—also produced under the "direction" of the regulator gene—are initially inactive and incapable of inhibiting the functioning of the operator gene and, by extension, the entire operon; however, they acquire this ability upon forming a complex with the end product or one of the End products of the biosynthetic process (Fig. 6.48). The end product thus acts as a corepressor. These insights were obtained through investigations of the Tryptophan and Histidine operons.

When E. coli cells grow on a medium containing ammonium salts as a nitrogen source, they are forced to synthesize all nitrogenous compounds and possess the full Complement of enzymes required to synthesize 20 Amino Acids. Upon the addition of a single amino acid (such as tryptophan or histidine) to the culture medium, the cell halts the synthesis of the entire set of enzymes necessary to produce these amino acids. This phenomenon is known as end-product (tryptophan or histidine) Repression of the enzymes catalyzing sequential Reactions of the corresponding metabolic pathway. The Regulation of the his and trp operons operates as follows: in the absence of histidine or tryptophan in the medium, the regulatory repressor protein lacks affinity for the operator, allowing the synthesis of the enzymes that catalyze the formation of these amino acids. Upon the addition of, for example, histidine (acting as a corepressor), conformational changes occur in the repressor protein molecule that has bound it, resulting in a significantly increased affinity of the entire complex (repressor protein-corepressor) for the operator. This ultimately halts the Transcription of the operon.

Evidence suggests that the corepressors in the synthesis of Amino acid metabolism enzymes may include not only free Amino acids as the end products of biosynthetic reactions, but also aminoacyl-tRNAs.

The regulation of structural Gene Expression involves the specific participation of CRP proteins (cAMP receptor protein), also known as CAP (catabolic gene activator protein). Upon interaction with cAMP, they form a complex that facilitates the binding of RNA polymerase to the promoter region and the initiation of operon transcription. Thus, CRP-cAMP serves as an additional regulator of synthetic processes within the cell. Operons sharing common regulators are termed regulons. Prokaryotic regulons also encompass genes encoding heat Shock proteins and genes involved in the SOS Response to DNA damage. Consequently, the repression and induction of protein synthesis in prokaryotes facilitate adaptation to changing environmental conditions.

The regulation of protein biosynthesis in eukaryotes is considerably more complex. Transcription and Translation are spatially separated (due to the presence of the nuclear membrane) and temporally segregated, which is of fundamental importance for the functioning of regulatory systems. Additional distinctive features of Eukaryotic cells include the structured nature of their genetic material: DNA is incorporated into Chromatin (in close contact with Histones and non-histone proteins), which must undergo complex structural modifications in transcribed regions prior to Transcription initiation; furthermore, genes are interrupted by introns, necessitating RNA splicing. Although the transcription initiation stage, alongside other complex steps of this process, represents a crucial checkpoint in the regulation of protein biosynthesis, post-transcriptional processes—at the levels of Processing, transport, and translation—are even more fundamental.

At the translational level, three primary modes of regulation can be distinguished. The first is positive regulation based on the affinity of mRNA for the initiation ribosome complex and initiation factors (mRNA discrimination). The second is negative regulation mediated by repressor proteins which, by binding to mRNA, block initiation (translational repression). These two mechanisms regulate individual mRNAs, meaning that the translation of each mRNA can be specifically controlled independently of other cellular mRNAs. The third mechanism involves Global Regulation of the Translation of the entire pool of cellular mRNAs through the modification of initiation factors.

The rate or frequency of Translation initiation by ribosomes can vary among different mRNAs. In eukaryotic cells, mRNA discrimination is driven by the differing affinities of initiation factors—rather than the ribosomes themselves—for distinct 5'-terminal initiation structures

of the mRNA. Initiation factors localize to small ribosomal subunits, which determines the varying efficiency of ribosome recruitment on different mRNAs, thereby categorizing them into "strong" and "weak" mRNAs. The distinct "strength" of an mRNA largely dictates the relative production levels of various cellular proteins. For instance, structural Membrane Proteins, ribosomal proteins, elongation factors, viral coat proteins, and other proteins required in large amounts are encoded by strong mRNAs, whereas many specialized enzymes and regulatory proteins are encoded by weak mRNAs. Therefore, mRNA discrimination can be viewed as a mechanism of constitutive control maintaining a fixed ratio of protein synthesis products.

A typical mechanism of translational repression involves a specialized protein, known as a repressor, specifically interacting with the region of the mRNA responsible for binding the ribosomal subunit during translation initiation. The repressor protein hinders the binding of the initiation ribosome complex, thereby either reducing the rate of initiation or completely blocking it.

In addition to conventional translational repression, eukaryotes have evolved an intriguing mechanism known as mRNA masking, whereby a given mRNA becomes inaccessible not only to translation initiation but is also effectively shielded from all other cellular processes—such as nuclease degradation and enzymatic modification of its 3'-end via polyadenylation. Masking is mediated by proteins and is regulated by external signals (effectors). The processes of mRNA masking and unmasking are particularly characteristic of gametogenesis (oogenesis and Spermatogenesis), early embryonic development, cellular differentiation, and hormonal regulation.

The most thoroughly studied pathway of global regulation of protein synthesis in eukaryotes is the activation of protein Kinases that phosphorylate the eIF-2 initiation factor, leading to the inhibition of translation initiation for all cellular mRNAs. Signals triggering their activation include heat shock and Other types of stress, growth factor deprivation, amino acid starvation, iron deficiency, and viral infections. The degree of protein synthesis inhibition can vary depending on the severity of the stress. Consequently, the numerous and interconnected pathways of translational regulation serve to coordinate all Stages of the process and finely tune the rate of protein synthesis.