Molecular Biotechnology: Principles and Applications - Glick, B., & Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
DNA, RNA, and Protein Synthesis
Deciphering Genetic Information; RNA and Protein

The vast majority of genes encode information for Protein Synthesis. Proteins are biological molecules involved in virtually all processes occurring in living systems. They serve as catalysts for diverse biochemical reactions, transport substances within and between Cells, regulate Cell membrane permeability, and form various structural elements. Proteins participate in motor Functions, provide defense against infections and toxins, and regulate the Synthesis of Other Gene products. The primary structural units of proteins are Amino Acids. All amino acids share a similar chemical Structure. Attached to the central carbon atom (the α-carbon) are a hydrogen atom (H), an amino group (NH₃⁺), a carboxyl group (COO^-), and an R-group (side chain) (Fig. 3.7, A). There are 20 different side chains and, accordingly, 20 amino acids. For instance, in The amino acid Alanine, the R-group is a methyl group (CH₃). Table 3.1 lists the one- and three-letter designations for amino acids. Connected to each other by peptide bonds, amino acids form a polypeptide chain. A peptide bond is formed between the carboxyl group of one Amino Acid and the amino group of another (Fig. 3.7, B). The first amino acid of a protein molecule has a free amino group (N-terminus), while the last has a free carboxyl group (C-terminus).

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Fig. 3.6. (Continued) B. A phosphodiester bond is formed between the α-phosphate group of the incoming nucleotide and the 3'-hydroxyl group of the growing chain. The next deoxyribonucleoside triphosphate is ready for complementary pairing.

Protein molecules range in length from 40 to over 1,000 amino acid residues, adopting various shapes (configurations, Conformations) depending on their sequence and Amino Acid Composition. Many functionally active proteins consist of two or more polypeptide chains (subunits), either identical or slightly different. Furthermore, many proteins with key functions are complex Protein Complexes made up of numerous distinct subunits.

Fig. 3.7. Generalized structural formula of an amino acid and peptide bond formation. A. Amino acid. R — side group. B. Formation of a peptide bond between two amino acid residues with side groups R1 and R2.

Table 3.1. Amino Acids and their designations

An important "intermediary link" in translating Genetic information from the language of NUCLEOTIDES to the language of amino acids is ribonucleic acid (RNA), which is synthesized on specific DNA regions serving as templates in accordance with their nucleotide sequence. RNA is a linear polynucleotide molecule that differs from DNA in two respects. First, the monosaccharide in RNA is ribose, which contains two hydroxyl groups instead of one; these are attached to the 2'- and 3'-carbon atoms. Second, one of the four bases in RNA is uracil (U), which replaces thymine. Most RNA molecules are single-stranded, although they frequently contain mutually complementary regions that form double-stranded structures known as "hairpins" (Fig. 3.8). Base pairing occurs in the same manner as in DNA, except that A–U pairs form instead of A–T pairs.

Fig. 3.8. Introduction/11.html">Secondary structure of a hypothetical RNA molecule. Complementary bases are linked by Hydrogen Bonds. The sugar-phosphate backbone is not shown.

There are three MAIN TYPES OF RNA: Messenger RNA (mRNA), ribosomal RNA (rRNA), and Transfer RNA (tRNA). All of them play a crucial role in decoding genetic information. The synthesis of RNA on a DNA template is called METABOLISM/31.html">Transcription. In most prokaryotes, the transcription of all RNA types is carried out by the same RNA polymerase. In eukaryotes, mRNA, rRNA, and tRNA are transcribed by different RNA polymerases.

Transcription is in many ways similar to Replication. The template for RNA Synthesis is a specific region of one of the DNA strands. RNA polymerase copies this region by sequentially linking ribonucleotides via 3'–5' phosphodiester bonds in accordance with the complementarity rule (Fig. 3.9). During transcription, the newly synthesized RNA molecule detaches from the DNA, and the DNA double helix reforms. To ensure that only specific DNA segments are transcribed, certain signal sequences must exist to indicate where transcription begins (initiation) and where it stops (termination). The initiation signal is usually located upstream of the coding sequence, while the termination signal is located downstream. The DNA region preceding the transcribed gene is called the 5'-flanking sequence, and the region following it is called the 3'-flanking sequence. From a molecular perspective, a gene is a specific nucleotide sequence transcribed into RNA. The vast majority of transcribed DNA sequences are so-called structural genes, which direct the synthesis of mRNA. The final product of a structural gene is a protein. In prokaryotes, a structural gene is a continuous segment of the DNA molecule. Transcription begins with the binding of RNA polymerase to the promoter, after which the entire structural gene (coding region) is copied sequentially from the first nucleotide to the last, yielding a functional mRNA (Fig. 3.10). In eukaryotes, most structural genes consist of multiple discrete coding regions (exons) separated by non-coding regions (introns). Upon completion of eukaryotic structural gene transcription, introns are excised from the primary transcript by Enzymes, and the exons are spliced together end-to-end (splicing) to form a functional mRNA (Figs. 3.11 and 3.12). Typically, exons range from 150 to 200 nucleotides in length, whereas introns vary from 40 to 10,000 nucleotides. Very few eukaryotic structural genes lack introns entirely. Occasionally, mRNA splicing can occur via alternative pathways. For example, in one tissue, a functional mRNA may be produced by joining all exons of the primary transcript, whereas in another, a particular exon may be excised along with its flanking introns, generating a different functional mRNA. Due to Alternative Splicing, different Tissues can produce distinct products from the same structural gene (Fig. 3.13).

Fig. 3.9. Schematic representation of transcription. The arrow indicates its direction.

Fig. 3.10. Schematic representation of a prokaryotic structural gene. Shown are the promoter (p), the Transcription initiation site and its direction (horizontal arrow), and the transcription termination region recognized by RNA polymerase (t). First, mRNA is synthesized on the DNA template (transcription), followed by the Synthesis of the protein chain (Translation).

Fig. 3.11. Schematic representation of a eukaryotic structural gene. Shown are the promoter (p), the transcription initiation site and its direction (horizontal arrow), and the transcription termination region recognized by RNA polymerase (t). 1–5 represent exons, while a–d represent introns. The primary transcript contains a poly(A) tail at the 3'-end and a methylated nucleotide G (cap) at the 5'-end. Following transcription, introns are excised from the primary transcript (Processing), and a protein molecule is synthesized on the resulting functional RNA (translation).

Fig. 3.12. Splicing of the primary transcript in eukaryotes. Angular arrows indicate the joining sites of exons 1, 2, and 3 after the removal of introns a and b.

In an actively functioning cell, approximately 3–5% of the total RNA accounts for mRNA, 90% for rRNA, and 4% for tRNA. mRNA can be represented by dozens of different molecular types, whereas rRNA is represented by only two types. The larger rRNA forms a ribonucleoprotein complex with proteins, known as the large ribosomal subunit, while the smaller rRNA forms a complex known as the small ribosomal subunit. During protein synthesis, the subunits combine to form a ribosome. In eukaryotes, both ribosomal subunits are larger than in prokaryotes. In addition to thousands of Ribosomes, a cell actively synthesizing proteins contains up to 60 Different types of tRNA. tRNA is a linear single-stranded molecule ranging from 75 to 93 nucleotides in length. It contains several mutually complementary regions that pair with one another (Fig. 3.8), and the overall molecule folds spatially into an L-shaped structure (Fig. 3.14). Specific enzymes (Aminoacyl-tRNA synthetases) attach the corresponding amino acid to the 3'-end of the tRNA. For example, the enzyme arginyl-tRNA synthetase attaches the amino acid arginine to the tRNA^Arg molecule. For each of the twenty amino acids that make up all proteins, there is at least one specific tRNA. At the other end of the tRNA molecule lies a three-nucleotide sequence called the anticodon. It recognizes a specific codon in the mRNA and determines which amino acid will be added to the growing polypeptide chain.

Fig. 3.13. Alternative splicing of the primary transcript in eukaryotes. Arrows indicate the exon-joining sites after intron removal. Exon 2, flanked by introns 1 and 2, is excised from the primary transcript, and exons 1 and 3 are joined to form a functionally active mRNA.

Fig. 3.14. Conformation of tRNA "loaded" with an amino acid. The dashed rectangle indicates the anticodon.