Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
The Eukaryotic Chromosome and Its Control
Poly (ADP-ribose)

An unusual component of The Cell nuclei in all higher organisms is formed by the polymerization of ADP-ribosyl groups derived from NAD⁺. A specialized enzyme catalyzing this polymerization process displaces nicotinamide and forms a glycosidic bond between the C-1 atom of ribose—to which the nicotinamide was attached—and the C-2' atom of the ADP component in the next monomer unit. The Physiological Role of poly(ADP-ribosyl groups) remains unclear at present, yet it has been found that they are covalently bound to Nuclear Proteins as well as to cytoplasmic components [304, 305] (Box 15-E).

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Box 15-E

Toxic Proteins; Diphtheria Toxin^a

Before an effective vaccine became available, infections with Corynebacterium diphtheriae caused one of the most feared childhood diseases. Although the pathogen merely produced superficial membranes in the throat, patients frequently died from severe systemic damage to multiple Organs. This was found to be caused by a potent heat-labile protein toxin. It was also discovered that the Bacteria produce this toxin only when infected with a temperate bacteriophage^b carrying the tox Gene, and provided that the concentration of inorganic iron in the medium is significantly reduced.

Diphtheria toxin is a protein with a Molecular Weight of approximately 62,000. Its minimum lethal dose for a guinea pig is a mere 0.16 mg/kg. Cell culture studies have shown that the toxin blocks amino acid incorporation into proteins by inactivating elongation factor EF-2, which is required for translocation in mammalian Ribosomes. The toxin acts similarly to an enzyme that transfers an ADP-ribosyl group from NAD⁺ to the EF-2 factor:

The modified EF-2 factor reacts with GTP in the usual manner, but the resulting complex is incapable of participating in translocation. To drive this fatal reaction, a cytoplasmic toxin concentration of only 10^-8 M is sufficient.

How does a protein toxin of this type penetrate the cell? There is reason to believe that a specific region of the protein molecule has The ability to bind to particular sites on The cell membrane. It is possible that binding at these sites stimulates pinocytosis. The toxin becomes active only after partial proteolytic Cleavage.

What is the Water/144.html">Origin of the tox gene, and why is it carried by a virus? Pappenheimer and Gill suggested that this gene somehow originated from a Introduction/5.html">Eukaryotic Cell gene encoding a functional protein. This gene integrated into the virus and, over the course of evolution, transformed into a gene determining the synthesis of a protein toxin. The presence of poly(ADP-ribose) in the Cell Nucleus (Section E, 3) suggests one possible origin for the tox gene. NAD⁺ serves as a substrate in the synthesis of this nuclear polymer, and the synthetase catalyzes the Cleavage of the ribosyl-nicotinamide bond, forming a new glycosidic bond between the C-1 of ribose and the 2-hydroxyl group of adenosine in the next monomer unit. It is conceivable that the synthetase gene itself was modified and transformed into the diphtheria toxin gene.

The group of bacteria-toxic proteins known as colicins was already discussed in Section G, 7. These also appear to bind to specific receptors on the outer membrane of bacteria such as E. coli. Neilands and his coworkers discovered that the colicin M receptor in E. coli also serves as a receptor for the siderochrome peptide ferrichrome (Box 14-B) and for bacteriophage T5. The antibiotic albomycin binds to this same membrane region. It has been hypothesized that early in evolution, bacteria developed molecules capable of forming chelate complexes with iron; these complexes gradually increased in size until they lost the ability to diffuse through the outer membrane into the cell. As a result, specific transport systems emerged, which were later exploited by phages and colicin-producing strains^c.

^a Pappenheimer, A. M., Jr., & Gill, D. M. (1973). Science, 182, 353–364.

^b Eklund, M. W., Poysky, F. T., Reed, S. M., & Smith, C. A. (1971). Science, 172, 480–482.

^c Lackey, M., Wayne, R., & Neilands, J. B. (1975). BBRC, 64, 687–693.

Questions and Problems

1. How can it be proven that hereditary traits are indeed encoded by The nucleotide sequence of DNA?

2. Describe the DNA Structure model proposed by Watson and Crick. Which biological and chemical facts can be explained using this model?

3. Provide experimental Evidence for the antiparallel orientation of strands in double-stranded DNA. Characterize the Specificity of the Enzymes used to obtain this evidence.

4. If all the DNA molecules in your body were joined into a single continuous Watson-Crick double helix, what would its length be?

5. Describe the Three types of RNA involved in Protein Synthesis. What is currently known about the chemical and physical properties, nucleotide composition, and Biosynthesis of each?

6. The lac Operon repressor protein contains only two Tryptophan residues, located at positions 190 and 209. Upon binding the inducer isopropyl-β-D-thiogalactoside, a small (~3 nm) yet reliable red shift occurs in the maximum of the protein's fluorescence spectrum. By introducing nonsense Mutations and suppressing them with appropriate suppressor genes, modified repressors were obtained (see Section G, 5; Sommer, H., Lu, P., & Miller, J. H., JBC, 251, 3774–3779, 1976). When Trp-190 was replaced by Tyr, the fluorescence spectrum shift upon inducer binding remained the same; however, when Trp-209 was replaced by Tyr, the shift was no longer observed. Comment on these results. Can you propose two possible explanations? How might one determine which of them is correct?

7. A bacterial ribosome with a diameter of 23 nm consists of 35% protein by weight. Assuming that proteins account for 35% of the volume, approximately how many protein molecules with an average molecular weight of 17,300 can be contained within the ribosome? (Assume maximal close packing; see also Table 15-5.)

8. During their synthesis and functioning, Transfer RNA molecules interact specifically with several enzymes. Write the equations for three reactions in which an enzyme recognizes a tRNA.

9. Name Three Functions of specific aminoacyl-tRNAs that are unrelated to their role in protein synthesis.

10. The Glycine-specific tRNA of E. coli contains the anticodon GCC. On the ribosomes, it can pair with either the GGU or the GGC codon. How is the ability of a single tRNA to recognize multiple different codons explained today?

11. A portion of an mRNA molecule with the following base sequence was isolated:

How will the corresponding peptide, encoded by this mRNA, be affected by a mutation that replaces the base indicated by the arrow with C?

12. The 5'-ends of many mRNA molecules feature long sequences that are not translated by the ribosomal protein-synthesizing system. Describe an experiment that would allow you to determine THE START OF the coding region in an mRNA molecule, assuming you have a pure preparation of a specific mRNA.

13. 7-Methylguanosine 5^,-monophosphate inhibits Protein synthesis in a cell-free system isolated from reticulocytes (Shafritz D. A. et al., Nature (London), 261, 291—294, 1976). The 5'-ends of most eukaryotic mRNA molecules contain 7-methylguanosine. However, the chemical removal of this group from vesicular stomatitis virus mRNA does not prevent its Translation in a reticulocyte cell-free system (Rose J. K., Lodish H. F., 262, 32—37, 1976). Comment on The Significance of these observations.

14. Define and describe the significance for biochemical genetics of each of the following terms:

15. Compare the following terms used in biochemical genetics:

16. Describe the following forms of DNA (or DNA-containing particles):

17. How can a piece of foreign DNA be inserted into a plasmid? Why does the incorporation of eukaryotic DNA into a plasmid carry both biochemical and ethical implications? What major biochemical problem might be solved in the future using this method?

18. How can heteroduplex analysis be used to determine the chromosomal Location of inserted foreign DNA?

19. a) What are the differences between E. coli and mammalian Chromosomes? How can these differences be detected? b) Define euchromatin, heterochromatin, satellite DNA, DNA repeats, and Histones. Indicate their putative functions.

20. Define the following types of mutants:

21. Describe the chemical Mechanisms of action of the following mutagenic substances or agents:

22. What specific properties would you predict for a Tyrosine tRNA with the GψA anticodon if a mutation converted it into a tRNA with the CψA anticodon?

23. When Mitochondrial DNA isolated from normal maize was digested with a restriction endonuclease and the resulting fragments were separated by agarose gel Electrophoresis, about 50 bands were detected on the electropherogram. In electrophoretic patterns obtained from a similar analysis of mitochondrial DNA from male-sterile maize (Sec. D, 9, d), certain bands present in normal mitochondrial DNA were missing (Levings C. S., III, Pring D. R., Science, 193, 158—160, 1976). Comment on this observation, as well as on the fact that when normal maize is crossed with male-sterile maize, the mitochondrial DNA pattern of the progeny always matches that of the maternal parent.

24. How would you explain the fact that a hinny has a milder temperament than a mule?