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

Molecular Genetics and Regulatory Systems
Molecular Genetics
Gene Expression Processes

We already know that a DNA molecule is a very long double helix consisting of two strands. Both strands are polymers built from four NUCLEOTIDES, which differ from one another by The Nature of their nitrogenous bases [adenine (A), guanine (G), cytosine (C), and thymine (T)]. The two strands in a DNA double helix have complementary sequences, such that A in one strand always pairs with T in the other; G and C pair in a similar manner.

A Gene is defined as a segment of DNA that encodes a specific protein. Gene Expression, which results in The formation of the Amino Acid Sequence of the corresponding protein, is a two-stage process; the intermediate product of this process is messenger ribonucleic acid (mRNA). In The First stage of gene expression, known as METABOLISM/31.html">Transcription, the complex oligomeric enzyme RNA polymerase catalyzes the synthesis of mRNA using the gene as a template. In the second stage, a peptide chain with the corresponding amino acid sequence is synthesized based on the information contained in The nucleotide sequence of the mRNA. This process, called Translation, involves various cellular components, including the centers and regulators of Peptide Synthesis—Ribosomes—as well as several different Transfer RNAs (tRNAs), which deliver chemically activated Amino Acids to the site of peptide chain synthesis in accordance with the sequence dictated by the mRNA. Below we examine these processes, as well as The Genetic Code, which serves as The Link Between the nucleotide and Amino acid sequences. In our Structure/133.html">Discussion, we will focus only on the main points and omit, where necessary, details that are significant in certain situations; therefore, the reader is strongly encouraged to explore The problem of GENE EXPRESSION IN more detail using the literature listed at the end of the chapter.

Transcription begins with the binding of the enzyme RNA polymerase to a signaling nucleotide sequence, known as a promoter, located on one of the complementary DNA strands. The nucleotide strand bound to RNA polymerase thus serves as the DNA template for the Synthesis of the mRNA molecule. After the DNA double helix partially unwinds near the RNA polymerase–promoter complex, the polymerase begins to move along the DNA template strand in the 3'→5' direction. When building the complementary DNA sequence of the single-stranded mRNA molecule, the rules of DNA–RNA base pairing are observed (recall Section 2.3.2 or see Fig. 6.1). The synthesized mRNA is antiparallel to the DNA template strand; consequently, mRNA is synthesized in the 5'-to-3' direction. In the example shown in Fig. 6.1, the lower DNA strand acts as the template. Transcription ceases—that is, mRNA synthesis stops—the moment RNA polymerase reaches a specific nucleotide sequence on the template strand (the terminator) that signals the end of synthesis. Before moving on to the next stages, it should be noted that RNA polymerase also binds non-specifically to DNA at sites that are not promoters. Non-specifically bound polymerase neither initiates nor performs transcription.

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FIG. 6.1. Schematic representation of the transcription of a DNA template strand (here, the lower strand is transcribed) by the enzyme RNA polymerase, resulting in the synthesis of a corresponding mRNA molecule with a complementary nucleotide sequence.

The length of an mRNA molecule ranges from approximately 300 to 3,000 nucleotide residues or more. Typically, one mRNA corresponds to a single gene in the DNA chain, but in some cases, an mRNA molecule carries Genetic information for a group of related and closely linked genes—the so-called Operon (see Section 6.1.4). As we noted in our discussion of Biosynthesis energetics in Chapter 5, the construction of an RNA chain proceeds via the Cleavage of a pyrophosphate group from nucleoside triphosphates. Consequently, the formation of each phosphodiester bond in RNA is accompanied by the cleavage of two high-energy phosphate bonds. This drives the Condensation reaction to completion and underscores The Importance of the synthesis of these Biopolymers for normal cellular function.

Before describing the translation process, we need to examine transfer RNAs in somewhat more detail. It is known that each type of tRNA can transport only one corresponding amino acid. Figure 6.2 shows the Introduction/11.html">Secondary structure of alanyl-tRNA from Yeast. The attachment of the alanyl residue to its respective tRNA requires the prior activation of Alanine by its conversion to alanyl-AMP, as reflected in reaction equation (5.39). The activated alanine residue is then attached to the appropriate tRNA with the participation of an enzyme specific for both The amino acid and the tRNA. This ensures that alanine is attached specifically to alanine tRNA.

We can now proceed to discuss the nature of tRNA Specificity. At the Base of the lower loop of the tRNA molecule, There is a sequence of three nucleotides called the anticodon. This structural element is present in all studied tRNAs. The base sequence of the anticodon is complementary to a three-nucleotide segment of mRNA called the codon. The Biochemistry of Nucleic Acids has shown that each codon represents a "word" in the genetic message—that is, each codon corresponds to a single amino acid. Since the chemical alphabet of RNA consists of only four letters (four bases: A, C, G, and U) and there must be at least 20 "words" (corresponding to the number of amino acids), a special "language" or code must exist for the transmission of genetic information. Surprisingly, the genetic code has been completely deciphered; furthermore, it has proven to be virtually universal across All living organisms—Bacteria, plants, and animals (Fig. 6.3).

The translation process begins with the binding of the smaller of the two ribosomal subunits to the mRNA molecule, mediated by a specific sequence called the ribosome-binding site. This site is usually located at a specific distance—which differs in PROKARYOTES AND EUKARYOTES—"upstream" of the AUG codon, where reading of the polypeptide chain sequence almost always begins. Here, the term "upstream" means that the site is located in the 5'-direction relative to the AUG codon. The sequence of codons that determines the amino acid sequence of the protein chain starts with the AUG codon and proceeds in the 5'→3' direction.

FIG. 6.2. The anticodon of alanine tRNA recognizes the complementary three-base codon in mRNA. (Watson, J., Molecular Biology of the Gene.)

Next, the mRNA forms a complex with the entire ribosome, and tRNAs carrying amino acids corresponding to the first two codons of the mRNA join this complex. In accordance with the specified amino acid sequence, a peptide bond is formed between the carboxyl group of the first Amino Acid and the amino group of the second. The ribosome then shifts in the 3' direction by one codon, releasing the tRNA of the first amino acid; subsequently, the third amino acid, in the form of the corresponding aminoacyl-tRNA, binds to the complex and attaches to the peptide chain, elongating the latter by one more residue. This process of Polypeptide chain elongation (Fig. 6.4) continues until a stop codon is reached (see Fig. 6.3); at this point, the peptide chain is released, and the ribosome–mRNA complex dissociates. Thus, the Construction of a polypeptide chain begins with the N-terminal residue and ends with the C-terminal amino acid residue.

FIG. 6.3. The genetic code. Three codons (Stop) signal the termination of peptide chain synthesis. It is easy to see that the substitution of a single base in a codon (especially the third one) often leads to a chemically similar amino acid (The Wobble Hypothesis / redundancy principle).

In Fig. 6.5, the translation process is depicted from a different perspective; here, the main focus is on The transfer of information from the nucleotide sequence of the mRNA to the corresponding amino acid sequence of the polypeptide. The hypothetical mRNA produced As a result of transcription (Fig. 6.1) is translated into the corresponding polypeptide. Note that here the trinucleotide sequence AUG fulfills two Functions: first, it indicates THE START OF the coding sequence for the protein, and second, it encodes Methionine (Met) as the N-terminal amino acid residue of the polypeptide. Note also that not the entire mRNA molecule carries information about The structure of the synthesized protein. As mentioned above, preceding the coding region is the ribosome-binding site; other nucleotide sequences, including coding sequences with their own initiation and termination codons, may be located downstream (in the 5' → 3' direction) of the coding region.

FIG. 6.4. Translation—the realization of mRNA genetic information into the Primary Structure of a protein—is carried out through a series of specific interactions between mRNA and tRNA on the ribosome. (Reproduced from: Edwards, N. A., Hassall, K. A., Cellular Biochemistry and Physiology, p. 342, McGraw-Hill Publishing Company Ltd., London, 1971.)

FIG. 6.5. Scheme of the transfer (translation) of information contained in the nucleotide sequence of an mRNA into the amino acid sequence of the corresponding polypeptide on the ribosome. The mRNA depicted here is the same as in Fig. 6.1.

At this point, it is useful to examine gene expression also from the standpoint of the elements that regulate the process, since controlling these elements is essential for successfully addressing Genetic Engineering tasks. Figure 6.6 illustrates in general terms the key functions of regulatory sequences in the transmission of genetic information. Upstream and downstream of the gene are promoter and terminator sequences that control the initiation and termination of transcription. The DNA must also contain translational regulatory signals (the ribosome-binding site, initiation codon, and termination codon) that are transcribed into mRNA. Finally, the portion of the mRNA complementary to the structural gene of DNA—which encodes a specific amino acid sequence—must be used to direct Protein Synthesis during translation.

Often, a single mRNA molecule binds to multiple ribosomes. Polyribosomes (Polysomes), which are complex aggregates of several individual ribosomes, frequently exhibit a highly ordered structure. In polyribosomes, genetic information is read simultaneously from various Regions of the mRNA, which significantly accelerates PROTEIN SYNTHESIS AND ensures high efficiency of this process.

Both mRNA, tRNA, and ribosomal Ribonucleic Acids (rRNA) are synthesized on a template—a specific region of a DNA strand. Thus, DNA contains encoded information not only on the Primary Structure of Proteins, but also on the architecture of the various molecules through which Protein synthesis is carried out.

FIG. 6.6. Regulatory and informational sequences of DNA direct the processes of transcription and translation (gene expression).

Here it is useful to recall that interactions between the amino acid residues of a polypeptide chain determine the secondary, tertiary, and even quaternary structures of proteins (Section 2.4). Consequently, weak interactions between atoms and groups play a major role at all stages of genetic information storage, transmission, and expression.

Before moving on to the Mechanisms of Genetic information transmission from generation to generation and the factors disrupting this process, we should mention the differences in protein synthesis mechanisms between eukaryotes and prokaryotes. In bacterial Cells, mRNA synthesis and its translation into proteins on ribosomes occur in virtually the same region of the nucleoid. In eukaryotes, transcription takes place in The Nucleus. Subsequently, mRNA molecules undergo modification (described in the following section), diffuse through the nuclear membrane pores into the Cytoplasm, and form complexes with ribosomes, where translation takes place. In eukaryotes, and to a lesser extent in prokaryotes, the translation product frequently undergoes modification resulting in the final, functionally active protein molecule. Post-translational modifications of proteins are discussed in Section 6.1.3. Finally, it should be noted that in both eukaryotes and prokaryotes, The process of protein synthesis also differs temporally; we will examine this issue in more detail in Section 6.5.