BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 29. EUKARYOTIC CHROMOSOMES AND GENE EXPRESSION IN EUKARYOTES

29.26. Thalassemia: A Genetically Determined Disorder of Hemoglobin Synthesis

The Study of Hemoglobin has contributed immensely to our understanding of protein Structure and function (Chapters 4 and 5). Similarly, investigations into hemoglobin genes and their expression have served as a vital source of insight into the functioning of eukaryotic genes. During embryonic development, embryonic Hemoglobins are successively replaced by fetal hemoglobin (HbF, α₂γ₂) and then by adult hemoglobin (HbA, α₂β₂). In addition, a small amount of HbA₂, which has the subunit structure α₂δ₂, is produced in adults. Recall that HbF has a higher oxygen affinity than HbA because it binds bisphosphoglycerate less tightly (Section 4.7). This increased oxygen affinity facilitates The transfer of oxygen from the maternal Circulation to the Fetal circulation. In reality, HbF is a mixture of two variants: one contains Glycine at position 136 of the γ chain, whereas the other contains Alanine. These variants are designated Gγ and Aγ, respectively.

All hemoglobin genes have been mapped. The haploid genome contains two tightly linked a-globin genes. These genes appear to be identical in all individuals, with the exception of rare mutants. Other globin genes are grouped in clusters on a different chromosome in the following order: Gγ-Aγ-δ-β (Fig. 29.37). It is interesting to consider why these functionally related genes are located adjacent to one another on the same chromosome. It is quite possible that their close proximity is required for switching expression from γ genes to δ and β genes during development. Alternatively, the linkage of these genes may reflect their evolutionary history. Most likely, the γ, δ, and β genes share a common ancestor that underwent tandem duplications followed by divergence.

Class="center">Fig. 29.37. Gene map of human γ, δ, and β globins

Thalassemias are a group of inherited anemias characterized by a reduced rate of synthesis of one of the hemoglobin chains. The term "thalassemia" is derived from the Greek word for "sea," reflecting the high prevalence of the disease among populations of Mediterranean origin. Major and minor thalassemias are observed in homozygous and heterozygous patients, respectively. The letter designation a or β indicates which chain is synthesized at a diminished rate. Today, studies of globin mRNA and cloned globin DNA are beginning to shed light on the causes of these disorders, and the molecular mechanisms underlying several types of thalassemia have been elucidated.

1. Gene deletion. In certain forms of a-thalassemia, one or both a-globin genes are deleted.

2. mRNA instability. In hemoglobin Constant Spring, the a chain contains 172 residues instead of 141 due to a mutation that converts the UAA stop codon into a Gln codon (CAA). Translation of a region that is normally noncoding somehow renders the mutant mRNA vulnerable to nuclease action.

3. Impaired chain initiation. In some types of β-thalassemia, Translation initiation proceeds too slowly, likely due to a defect in the 5'-untranslated region.

4. Premature chain termination. One form of β-thalassemia arises from a single-base substitution in the Lys codon AAG, generating a UAG stop codon at position 17.

5. Reduced mRNA production. In many forms of β-thalassemia, the β-globin gene is present, but very little β-globin mRNA is produced. The underlying cause of this phenomenon is currently under intense investigation. It is possible that in some types of β-thalassemia, intervening sequences are not excised properly¹.

29.27. Translation Is Regulated by a Protein Kinase Cascade That Inactivates an Initiation Factor

Reticulocyte lysates synthesize hemoglobin subunits at a high rate until heme is depleted. In the absence of heme, Protein Synthesis ceases due to the rapid formation of an enzymatic inhibitor of protein synthesis, which is a protein kinase. The target of this kinase is eIF-2, an initiation factor that binds GTP and delivers Met-tRNA_i to the 40S ribosomal subunit². Inactivation of eIF-2 by phosphorylation leads to a block in the initiation of protein synthesis. How does heme regulate The activity of this kinase? This effect is mediated by another kinase (Fig. 29.38). The eIF-2 kinase that modifies the initiation factor exists in two forms: an inactive dephosphorylated form and an active phosphorylated form. The phosphorylation of eIF-2 kinase is catalyzed by a cyclic AMP-dependent kinase composed of Two Types of subunits: two regulatory (R) and two catalytic (C). The inactive R₂C complex dissociates in the presence of cyclic AMP into two catalytically active C subunits and two R subunits. Heme blocks this dissociation process, preventing the activation of the two Kinases in the regulatory system. As a result, eIF-2 is not phosphorylated and retains its activity in initiating protein synthesis. This protein kinase cascade is reminiscent of the Regulation of Glycogen METABOLISM (Section 16.15). These processes also share the common feature that the regulatory action of these kinases is reversed by specific Phosphatases.

¹ In the case of so-called β⁺-thalassemia, this has indeed proven to be true, with the sole difference between the mutant gene and the wild-type gene being a single-nucleotide substitution within the intron. Translator's Note.

² The cascade regulatory mechanism described by the author and proposed in Ochoa's laboratory turned out to be incorrect. Although cAMP-dependent protein kinase phosphorylates many Proteins, it does not act on eIF-2 and has no effect on protein kinase activity under physiological conditions. Translator's note.

Fig. 29.38. Phosphorylation cascade inactivating the initiation factor eIF-2

29.28. Diphtheria Toxin Blocks Eukaryotic Protein Synthesis by Inhibiting Translocation

Before the advent of effective immunization, diphtheria was a major cause of childhood mortality. The lethal effect of the disease is caused primarily by a toxin produced by Corynebacterium diphtheriae, a bacterium that grows in the Upper Respiratory Tract. The structural gene for the toxin is localized in a lysogenizing phage carried by certain strains of C. diphtheriae. A few micrograms of this toxin, which has a mass of 61 kDa, typically constitute a lethal dose for an unimmunized human, as this amount is sufficient to inhibit protein synthesis. Diphtheria toxin blocks the elongation phase of eukaryotic protein synthesis by inactivating the elongation factor required for translocation. This factor, designated elongation factor 2 (EF-2) or translocase, performs a role in eukaryotes analogous to that of EF-G in Bacteria. Translocase is required for the GTP-dependent movement of peptidyl-tRNA from the A site to the P site and the concomitant movement of Messenger RNA immediately following peptide bond formation. The mechanism by which translocase is inactivated by diphtheria toxin is particularly interesting. The toxin catalyzes the Covalent Modification of translocase, utilizing NAD+ as a donor of the adenosine diphosphate ribose (ADPR) moiety, with the concomitant release of nicotinamide.

The diphtheria toxin molecule consists of two parts and can be cleaved into two fragments with masses of 21 kDa (fragment A) and 40 kDa (fragment B). Domain B binds to The surface of susceptible Cells, whereas domain A catalyzes the ADP-ribosylation of translocase. Specifically, domain B binds to Cell/30.html">The Plasma Membrane ganglioside G_M1, enabling the catalytic domain to enter The Cell. Upon entry, the bound toxin is cleaved such that fragment B remains on the cell surface while the hydrophilic fragment A is translocated into the Cytosol. Interestingly, many Other toxins, such as cholera toxin (Section 35.7), likewise consist of a cell-surface-binding domain and a catalytic domain that inactivates a crucial cellular component.

29.29. Ribosomes Associated with the Endoplasmic Reticulum Synthesize Secretory and Membrane Proteins

In Eukaryotic cells, some Ribosomes float freely in the cytosol, whereas others are bound to the extensive membrane system known as The Endoplasmic reticulum (ER). Regions of the ER studded with ribosomes are referred to as rough ER because they appear bumpy on electron micrographs (Fig. 29.39), in contrast to the smooth ER, which lacks ribosomes. Cells that secrete large amounts of protein, such as pancreatic acinar cells, feature a prominently developed rough ER. In general, all known secretory proteins are synthesized by ER-bound ribosomes. In addition, ribosomes associated with this membrane system synthesize many proteins destined for the plasma membrane and Organelles such as Lysosomes.

Fig. 29.39. Electron micrograph of the rough endoplasmic reticulum

Fig. 29.40. Electron micrograph of crystalline arrays of membrane-bound ribosomes in oocytes of hibernating lizards

Membrane-bound ribosomes in the oocytes of hibernating lizards form crystalline arrays (Fig. 29.40). These ordered structures are currently being investigated using three-dimensional image reconstruction techniques. A low-resolution map reveals that both the large (60S) and small (40S) subunits lie near the membrane surface. The large subunit possesses a projection that extends into the membrane (Fig. 29.41). Unlike the smooth ER, the rough ER contains two transmembrane proteins called ribophorins, which specifically interact with the large ribosomal subunit.

Fig. 29.41. Membrane-bound ribosome. This low-resolution image was obtained by reconstruction analysis from a series of Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF ordered ribosomes taken at various angles

Three fundamental questions arise regarding the synthesis and subsequent fate of proteins produced on ER ribosomes.

1. Are there two distinct classes of ribosomes—free cytosolic ribosomes and membrane-bound ribosomes—or are all ribosomes fundamentally identical? If there is only a single class of ribosomes, what determines whether a given ribosome remains free or binds to the rough ER?

2. How does the newly synthesized polypeptide chain emerging from a membrane-bound ribosome cross the permeability barrier of the rough ER? For example, secretory proteins such as pancreatic zymogens (Sec. 8.1) are found within the ER lumen shortly after synthesis.

3. What determines the ultimate fate of a protein synthesized on a membrane-bound ribosome? Some of these proteins are exported from the cell, whereas others are targeted to intracellular organelles. Furthermore, proteins synthesized by the rough ER are incorporated as integral Components of the plasma membrane and intracellular membranes.