Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

The Chemical Foundations of Life
Sugars and Polysaccharides
Storage of Biological Information, DNA and RNA

Like Polysaccharides, polynucleotides are formed by the Condensation of monomers. In both RNA and DNA, NUCLEOTIDES are joined by phosphate bonds formed involving the hydroxyl groups at C-3' and C-5' of ribose or deoxyribose. As an example, Fig. 2.10 shows The Structure of a trinucleotide. Cellular DNA molecules are incredibly large: all the hereditary information of prokaryotes is stored in a single DNA molecule with a molecular mass of about 2∙10⁹. In eukaryotes, The Nucleus may contain several large DNA molecules. The negative charges of DNA are neutralized by divalent cations (in prokaryotes) or basic Amino Acids (in eukaryotes).

It should be emphasized that The nucleotide sequence, as shown in Fig. 2.10, a, has a direction or polarity due to the fact that There is a free hydroxyl group at C-5' at one end of the chain, and a hydroxyl at C-3' at the other. Fig. 2.10, b shows a convenient way of representing a nucleotide sequence in the 5'-to-3' direction. Even shorter notations are often used. Suppose, for example, that bases 1, 2, and 3 in Fig. 2.10, b are cytosine, adenine, and thymine, respectively; then the same nucleotide sequence can be written as pCpApT or even shorter: CAT.

As proposed in 1953 by James Watson and Francis Crick, the DNA molecule consists of two polynucleotide chains forming a double helix (Fig. 2.11). The backbone of the molecule, which has the shape of a regular helix, is built of sugar residues and phosphate groups. Purine and pyrimidine bases are located inside The Double Helix. It is The sequence of four different nitrogenous bases in the polynucleotide chain that contains all the Genetic information, in particular, data on the structure of synthesized Ribonucleic Acids and Proteins. The mechanisms regulating and utilizing the genetic information of DNA will be discussed in Chapter 6.

The nitrogenous base of one chain of the double helix interacts with the spatially closest Base of the other chain in a strictly defined manner, so that adenine specifically binds only to thymine, and guanine to cytosine. Fig. 2.12 shows the remarkable geometric similarity of these Base Pairs, as well as the presence of two and three Hydrogen Bonds in the A–T and C–G base pairs, respectively.

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FIG. 2.10. a—condensation of several nucleotides to form a chain linked by phosphodiester bonds; b—simplified schematic representation of a nucleotide chain.

FIG. 2.11. The STRUCTURE OF THE DNA double helix represented in several ways. This figure shows the classic Watson-Crick structure [7], also called B-DNA; it is a right-handed helix in which the planes of the base pairs are perpendicular to the helix axis. At the bottom of the figure is a diagram illustrating some geometric parameters of this structure. [The upper and lower figures are reproduced with permission from: Yudkin M., Offord R., A Guidebook to Biochemistry, p. 52, Cambridge University Press, London, 1971.]

FIG. 2.12. Complementary base pairs adenine–thymine and guanine–cytosine, formed by Hydrogen bonds and having very close spatial characteristics, which facilitates The formation of base pairs inside the double helix of the DNA molecule. (From: Loewy A., Siekevitz P., Cell Structure and function. — Moscow: Mir, 1971, p. 164.)

The two DNA strands have opposite orientations (5'→3' and 3'→5'). Therefore, knowing the nucleotide sequence of one deoxyribonucleotide strand, for example, 5'CGAATCGTA3', one can deduce the structure of the corresponding fragment of the double-stranded DNA molecule; in our case, this fragment will have the structure

5'CGAATCGTA3'

3'GCTTAGCAT5'

We would arrive at the exact same double-stranded structure if we were given the sequence 5'TACGATTCG3'. Thus, in an informational sense, the sequence of one strand determines the sequence of the complementary strand, i.e., each strand serves as a template for the other.

This characteristic property of DNA is the basis for the synthesis of daughter DNA from the original parental DNA molecule—a process called METABOLISM/36.html">DNA Replication. If the two complementary DNA strands separate and then Double helices are built on each of the strands in accordance with the base-pairing rules, the final product of this process will be two new molecules, each identical to the original double-stranded DNA and containing one new and one old strand (Fig. 2.13). Thus, base pairing forms the chemical basis for reading the biological information encoded in the nucleotide sequence of DNA. The validity of the General Principles of this mechanism, schematically shown in Fig. 2.13, was convincingly proven by experiments in which E. coli Cells were grown first in a medium containing ¹⁵N and then in a medium with only the normal nitrogen isotope ¹⁴N. DNA molecules containing different isotopes can be separated in an ultracentrifuge. Ultracentrifugation and The Use of isotopic labels, including radioactive ones, are essential tools for experimental research in modern biochemistry.

Another important detail of Introduction/20.html">DNA Structure is the hydrogen bonds through which base pairs are formed (Fig. 2.12). Hydrogen bonds contribute to the stabilization of the double-stranded structure of DNA, but this stabilization is neither permanent nor irreversible. As shown in the replication diagram (Fig. 2.13), the biological function of DNA relies on the unwinding of the helix and the Separation of the two strands. In the course of further study of Molecular Genetics (Chapter 6), we will also become acquainted with other important Functions of single-stranded DNA segments.

The separation of the two DNA strands upon heating is The basis of an important method for identifying different DNAs. Since AT base pairs are held together by two hydrogen bonds, and GC pairs by three, DNA regions containing a larger number of AT pairs melt (as The process of strand separation is conventionally called) earlier than regions enriched in GC pairs. The melting process is easily monitored by measuring the absorbance of the DNA solution at 260 nm; single-stranded DNA absorbs more strongly, and the DNA melting process is recorded by an increase in total absorbance. The melting Temperature, T_m, is defined as the temperature at which The change in absorbance is 50% of the difference in absorbance between fully double-stranded and fully melted (single-stranded) DNA. As expected, the T_m value correlates with the GC content. For example, DNA from E. coli, containing 50% GC, has a T_m = 69°C, while DNA from Pseudomonas aeruginosa with 68% GC is characterized by a T_m = 76°C. The GC content in various organisms varies widely (from 23 to 75%). Features of nucleotide composition are sometimes used to identify organisms.

FIG. 2.13. Simplified scheme of DNA replication. As the strands of the parental DNA separate, complementary strands are synthesized on them, resulting in the formation of two daughter molecules identical to the parental DNA. Note that each daughter molecule contains one strand of the parental DNA. (From: Loewy A., Siekevitz P., Cell Structure and Function. — Moscow: Mir, 1971, p. 169.)

If a solution of melted DNA is cooled, the separated complementary strands reform a double helix structure (this process is called annealing). Similarly, two different single-stranded DNA segments with partially complementary sequences can undergo Hybridization to form a double-stranded segment. Hybridization is a crucial experimental technique in biochemistry and Recombinant DNA technology (Chapter 6). To conclude this brief Introduction to the physical chemistry of DNA, it should be noted that double-stranded DNA can also be melted or denatured by adding alkali or acid, which leads to The ionization of the bases. It is also worth mentioning that from a hydrodynamic standpoint, double-stranded DNA in solution behaves like a rigid rod, whereas single-stranded DNA behaves like a random polymer coil.

As mentioned above, all the information required for a cell to perform its essential functions, including growth and division, is contained in Bacteria like E. coli within a single DNA molecule that has the structure of a giant circle. In its Native State, E. coli DNA exists in a supercoiled form, which is clearly visible in Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF the bacterium as a somewhat diffuse nucleoid (see Fig. 1.2). This large, primary DNA of The Cell is called the bacterial chromosome; the circular chromosome of E. coli is built of 4.7 million base pairs*.

In the membrane-bound nucleus of eukaryotes, DNA is typically divided among several Chromosomes; the latter can be much larger than prokaryotic chromosomes. Eukaryotic chromosomes also include small basic proteins called Histones, which make up about half of the chromosome's mass. This complex of Nucleic Acids and Proteins in eukaryotic chromosomes is called Chromatin. Chloroplasts and Mitochondria of Eukaryotic cells also contain separate, smaller DNA molecules.

It is highly significant that cells can also contain other DNA molecules. Relatively small DNAs existing in many bacteria and eukaryotes are called Plasmids. Bacterial plasmids, example, are circular DNA molecules containing from 4 to 50 thousand base pairs. The BIOLOGICAL FUNCTIONS OF plasmids are to provide the host cell with useful, but not absolutely essential, traits, such as Antibiotic Resistance. In biochemical research and biotechnological processes, plasmids occupy a central place, serving as an important tool for recombinant DNA Methods. As we will see in Chapter 6, modifying plasmids in laboratory conditions and subsequently introducing recombinant plasmids into living cells allows their genetic program to be altered, forcing them to produce new compounds or grow more efficiently.

* DNA length expressed in kilobases can be converted into units of length, assuming that 1 kilobase corresponds to a length of 0.34 µm. In addition, the molecular mass of double-stranded DNA with 1 kilobase is approximately 660,000 atomic mass units.

Other small DNA molecules can be introduced into living cells by Viruses. Small viruses capable of infecting bacteria are called Bacteriophages or, for short, phages. The latter can cause A number of difficulties in large-scale biotechnological processes using pure bacterial cultures. At the same time, The Study of phages has made a major contribution to The Development of molecular genetics. In addition, phages are used to create stable working collections or libraries of DNA segments. Phage λ of E. coli contains a single circular double-stranded DNA molecule built of 48.6 thousand base pairs. The DNA of E. coli phage T2 consists of 166 thousand base pairs.

The primary function of DNA is to store instructions for the synthesis of RNA molecules of a specific nucleotide sequence and length; some of these RNAs then direct the synthesis of various specific proteins. The DNA segment encoding the sequence of an RNA molecule is called a Gene. Below, we will briefly review the Different types of RNA and then proceed to discuss proteins and the structural elements from which they are built.

All cells contain Three types of ribonucleic acids, while a fourth type is characteristic of certain viruses. RNA molecules are composed of ribonucleotide residues, whose carbohydrate component is ribose (rather than deoxyribose, as in DNA). Like DNA, nucleotides in RNA are arranged in a specific sequence. In essence, the nucleotide sequence of RNA is constructed based on the information contained in the corresponding DNA segments. In The transfer of information from DNA to RNA, base pairing again plays a central role. The rules of base pairing in this case are analogous to those governing base pairing in double-stranded DNA, with the exception of the adenine-thymine pair; during RNA Synthesis, an adenine-uracil pair is formed instead:

In the normal Cell Cycle, various RNAs participate, performing the crucial function of reading and expressing the genetic information stored in DNA. Messenger RNA (mRNA) is complementary to The base sequence of a gene in DNA. Each mRNA molecule carries specific information from DNA to another part of the cell's biochemical machinery. Since The amount of this information varies within a fairly wide range, the size of mRNA molecules can also vary; typically, an mRNA chain contains 10³—10⁴ nucleotides. RNAs are almost always single-stranded, whereas the formation of intermolecular and intramolecular base pairs often plays an important role in RNA Structure or function.

The information contained in mRNA is translated in the ribosome (Figs. 1.2 and 1.3). Up to 65% of the ribosome consists of ribosomal RNA (rRNA), which in turn can be separated (for example, by centrifugation) into several types of RNA. In particular, the E. coli ribosome contains three different RNAs, designated 23S, 16S, and 5S, which contain about 3∙10³, 1.5∙10³, and 10² nucleotide residues, respectively. In Ribosomes from the Cytoplasm of eukaryotic cells, on the other hand, there are 28S, 18S, 7S, and 5S rRNAs, while ribosomes of Mitochondria and chloroplasts contain RNAs specific only to these Organelles.

Transfer RNAs (tRNAs) have the lowest molecular weight and contain only 70 to 95 nucleotide residues; they are located in the Cell Cytoplasm and participate in the Translation of the Genetic Code in the ribosome. Table 2.5 lists the Main Properties of various RNAs of the bacterium E. coli. The nucleotide sequences and, consequently, the structures and functions of various cellular rRNAs and tRNAs, as well as mRNAs, are determined by the nucleotide sequences of the corresponding genes in cellular DNA.

The ultimate result of this complex process of information transfer and translation is the synthesis of a protein molecule. Proteins represent the tangible biochemical expression of the genetic information and instructions carried by DNA. Using terminology from process control theory, Proteins can be described as the final control elements that transmit DNA command signals to cellular processes. Below and in some subsequent chapters, we will examine the dynamics and properties of these control elements.

Table 2.5. Properties of E. coli RNA^a

"Reproduced from: Lehninger A. L. Biochemistry, 2nd ed., Table 12-3, Worth Publishers, Inc., New York, 1975; there is a translation of an earlier edition: Lehninger A., Biochemistry.— Moscow: Mir, 1976.