Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Gene Expression
Translation of the Genetic Code

Translation is The process of mRNA decoding, whereby the Genetic information encoded in The nucleotide sequence of an mRNA molecule is translated into the Amino Acid Sequence of a polypeptide chain. mRNA decoding proceeds in the 5' → 3' direction. The translational process comprises the following stages:

1) Amino Acid Activation;

2) tRNA aminoacylation;

3) translation proper.

Amino acid activation. This is the process wherein an amino acid is attached via its carboxyl group to the a-phosphate of ATP with the involvement of a specific aminoacyl-tRNA synthetase (Fig. 3.10). The reaction is accompanied by the release of inorganic pyrophosphate and The formation of aminoacyl-adenylate (AMP-AA). Aminoacyl-adenylate exhibits very high reactivity and is stabilized through tight binding to the enzyme. This process is characterized by high Specificity: each amino acid has its own dedicated enzyme or Enzymes.

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Fig. 3.10. Amino acid activation during translation

tRNA aminoacylation. This involves The transfer of the aminoacyl group from the enzyme-bound aminoacyl-adenylate to the 2'- or 3'-OH group of the terminal ribose of tRNA located in the acceptor stem (Fig. 3.11).

A key feature of the reaction leading to tRNA aminoacylation is the high Specificity of the enzymes involved. The attachment of each of the 20 Amino Acids found in Proteins to its corresponding tRNA is catalyzed by a specific aminoacyl-tRNA synthetase. The enzyme must distinguish one amino acid from the other 19 and transfer it to one or more isoacceptor tRNAs out of approximately 75 other available tRNA species. It is important to emphasize the striking structural similarity among many amino acids (such as leucine, valine, and isoleucine; valine and Threonine; aspartic and glutamic acids; etc.), as well as the remarkable resemblance in the secondary and tertiary structures of tRNAs. Consequently, even the exceptionally high specificity inherent in these enzymes is insufficient to completely prevent errors, and synthetases possess proofreading activity to correct mischarging. This occurs via the Hydrolysis of the bond between The amino acid and AMP within the enzyme–aminoacyl–adenylate complex, thereby preventing the formation of misaminoacylated tRNA. Conversely, there is no mechanism to remove an incorrect amino acid once it has already been attached to a tRNA. In such cases, the erroneous amino acid is incorporated into the protein. The frequency of these errors is very low (e.g., $10^{-5}$ in rabbit Hemoglobin).

Fig. 3.11. Formation of aminoacyl-tRNA

Translation proper. The process of translation takes place on Ribosomes—cellular Organelles that represent complex macromolecular assemblies composed of proteins and RNA molecules. Throughout the entire Protein Synthesis process, the growing polypeptide chain, the mRNA, and the incoming aminoacyl-tRNA remain attached to the ribosome. Ribosomes differ in size and composition between PROKARYOTES AND EUKARYOTES (Fig. 3.12). The sedimentation coefficient of prokaryotic ribosomes is 70S (where S stands for Svedberg, a unit measuring the rate at which a particle sediments during centrifugation; 1S = 10^-13 s), whereas cytoplasmic ribosomes in eukaryotes have a sedimentation coefficient of 80S.

Under certain conditions, ribosomes can dissociate into large and small subunits, and each subunit, in turn, into its constituent Protein and RNA molecules (Fig. 3.12). All of these components can reassociate to form a functionally active ribosome under appropriate conditions.

Fig. 3.12. Structure and composition of prokaryotic and eukaryotic ribosomes

Electron Cell/15.html">Microscopy studies of 70S ribosomes have demonstrated that the small and large subunits contact each other at several points, forming a cleft between them that accommodates the mRNA during translation. Two functionally important sites on the 70S ribosome are crucial for understanding the translation process: the A site (aminoacyl site), which binds the incoming aminoacyl-tRNA, and the P site (peptidyl site), which binds the growing peptide chain.

In addition to aminoacyl-tRNAs and ribosomes, translation involves A large number of accessory proteins known as initiation, elongation, and termination factors.

The core of the translational process consists of the sequential decoding of mRNA in the 5'→3' direction mediated by aminoacyl-tRNAs, accompanied by the successive Condensation of amino acid residues—starting from the amino (N) terminus of the polypeptide chain toward the carboxyl (C) terminus. The template principle is strictly observed through the recognition of complementary NUCLEOTIDES between the successive mRNA codon and the tRNA anticodon. Translation has been most thoroughly studied in prokaryotes, and The Mechanism of this process will be examined using E. coli as a model.

Translation initiation. Reading of the mRNA begins at the AUG codon, which marks the 5' end of the coding sequence and specifies the N-terminal (first) amino acid of the synthesized polypeptide. Initiation requires the 30S ribosomal subunit, which forms a complex with initiation factors (IF1, IF2, IF3), GTP, and fMet-tRNA. This pre-initiation complex binds to the 5' end of the mRNA coding sequence near the AUG start codon. Apparently, IF2 is able to distinguish fMet-tRNA (formylmethionine-tRNA) from Met-tRNA, which binds to internal AUG codons within the mRNA but cannot initiate translation from the start codon. This specificity is conferred by the N-formyl group, which is absent in Met-tRNA.

Recognition of the start codon occurs as follows. The binding of the 30S subunit to mRNA is tightly regulated by a nucleotide sequence located approximately 10 nucleotides upstream of the 5' end of the start codon. This interaction is facilitated by complementary base pairing between this purine-rich sequence and a polypyrimidine stretch within the 16S rRNA. The initiation process depends on various structural constraints of the interacting regions, including the Introduction/11.html">Secondary structure of the mRNA segment surrounding the AUG start codon, which plays a regulatory role in controlling protein synthesis efficiency.

Thus, during initiation, this complex binds to the P site of the 30S ribosomal subunit, making formylmethionine the first amino acid in the peptide chain. This is followed by the association of the 50S ribosomal subunit to form the 70S initiation complex (Fig. 3.13). The energy required to initiate Protein synthesis is derived from the hydrolysis of GTP to GDP and P_i.

Translation elongation. For the Formation of the first peptide bond, the aminoacyl-tRNA corresponding to the next codon must occupy the A site of the ribosome. To achieve this, the aminoacyl-tRNA must first bind the elongation factor EF-Tu and GTP. The resulting ternary complex (aminoacyl-tRNA·EF-Tu·GTP) delivers the aminoacyl-tRNA to the A site. During this process, GTP is hydrolyzed, and the (EF-Tu·GDP) complex dissociates from the ribosome. Once both the A and P sites are occupied, the peptidyl transferase activity of the 50S subunit catalyzes the transfer of the fMet group from its tRNA to the amino group of the aminoacyl-tRNA residing in the A site (Fig. 3.14). As a result, a dipeptidyl-tRNA is formed in the A site, while a deacylated (free) tRNA remains in the P site (Fig. 3.13).

The peptidyl transferase activity of ribosomes is believed to reside not in the protein moiety of the 50S subunit, but rather in one of its RNA components—specifically acting as a ribozyme.

Fig. 3.13. Translation of the Genetic Code: 1 — formation of the 70S initiation complex; 2 — binding of aminoacyl-tRNA to the ribosomal A site; 3 — peptide bond formation; 4 — ribosomal translocation. Steps 2–4 repeat during the elongation cycle

To read the next codon and extend the polypeptide chain by one more amino acid, the entire series of reactions must repeat. However, before this happens, the free tRNA vacates the P-site, the newly formed dipeptidyl-tRNA shifts to it from the A-site (without codon-anticodon interaction taking place), and the ribosome advances in a stepwise manner (by 3 nucleotides) toward the 3'-end of the mRNA. All these processes are driven by the elongation factor EF-G via GTP-dependent ribosome translocation. As a result of these three events, the A-site is freed and the next codon is exposed, allowing the subsequent elongation cycle to begin (Fig. 3.13). It should be noted that the formation of each peptide bond consumes energy equivalent to four energy equivalents (assuming the energy of a phosphate bond formation equals one equivalent): two equivalents of ATP are utilized during tRNA aminoacylation, and two equivalents of GTP are consumed in each elongation cycle.

Fig. 3.14. Formation of a peptide bond between the first Two amino acids on ribosomes

Translation termination. The process of sequential codon translation eventually reaches a point where one of the three stop codons—UAG, UAA, or UGA—appears in the A-site. Nature lacks tRNAs whose anticodons correspond to these codons. This is where termination factors RF-1 and RF-2 come into play, catalyzing the release of the polypeptide chain from the tRNA, the tRNA from the ribosome, and the 70S ribosome from the mRNA.

Following translation initiation, the 70S ribosome moves away from the initiation site as each subsequent codon is read. Once the distance from the ribosome to the initiation site reaches 100–200 nucleotides, a new initiation event can take place at that site. Furthermore, as soon as the second ribosome covers the same distance, a third initiation can occur, and so on. Thus, a single protein-coding mRNA sequence can be translated simultaneously by multiple ribosomes. Such multiribosomal translation complexes are called polyribosomes or Polysomes.

Messenger RNAs consisting of several protein-coding regions are often translated sequentially: when a ribosome reaches the stop codon in the first sequence, it dissociates from the mRNA, and a new complex binds to the next initiation site. Sometimes this does not happen, and the ribosome translating the first coding sequence continues to move along the mRNA without dissociating, initiating translation at other sites.

In some cases, the translation of the first coding sequence may begin and even finish before the METABOLISM/31.html">Transcription of the remaining sequences is complete, as, for example, in the case of the lac or trp operons in E. coli.

Features of eukaryotic translation. The process of eukaryotic mRNA Translation is generally similar to that of prokaryotes. However, there are several key differences. First, transcription and translation apparatuses in eukaryotes are separated both temporally and spatially, since transcription takes place in The Nucleus, whereas translation occurs in the Cytoplasm. Second, the initiating aminoacyl-tRNA in eukaryotes is not fMet-tRNA, but a specialized initiating Met-tRNA. Third, the 5'- and 3'-ends of eukaryotic mRNAs feature specific structures—caps and tails—that participate in translation. Notably, certain translation initiation factors recognize the capped regions to bind to the mRNA and initiate the translation process.