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

Fundamentals of Molecular Biotechnology
DNA, RNA, and Protein Synthesis
Translation

Translation is carried out with the participation of mRNA, various tRNAs "charged" with their respective Amino Acids, Ribosomes, and numerous protein factors that drive the initiation, elongation, and termination of Polypeptide chain synthesis. In Prokaryotic Cells, translation is initiated by formylmethionine tRNA, appropriately called the initiator tRNA. Assisted by protein factors, the 3'-UAC-5' anticodon of the initiator tRNKf^Met (where fMet denotes a modified Methionine with a formylated amino group) binds to the 5'-AUG-3' mRNA codon, which forms a complex with the small ribosomal subunit. No other tRNA is capable of binding to this complex. In turn, mRNA binds to the small ribosomal subunit through nucleotide pairing between an approximately eight-nucleotide sequence (the Shine-Dalgarno sequence) located near the 5'-end of the mRNA and the complementary 3'-end sequence of rRNA associated with the small ribosomal subunit. The large subunit then joins the fMet-tRNA^fMet-mRNA-small subunit complex to form the initiation complex (Fig. 3.15).

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Fig. 3.15. Translation initiation in a Introduction/4.html">Prokaryotic Cell. The Shine-Dalgarno (S-D) sequence, situated near the 5'-end of the mRNA, pairs with the complementary 3'-end sequence of the rRNA complexed with the small ribosomal subunit. The anticodon (UAC) of the initiator fMet-tRNA^fMet base-pairs with the start codon (AUG) of the mRNA. The large ribosomal subunit joins the resulting complex, establishing the initiation complex. The amino group of the methionine attached to the initiator tRNA is formylated (CHO) (not shown in the diagram). Following translation, the formylmethionine is cleaved from the protein chain.

In eukaryotes, translation is initiated by the binding of a specific "charged" initiator tRNA (Met-tRNA^Met) and initiation factors to the small ribosomal subunit. Subsequently, the mRNA attaches via its 5'-end to the tRNA-small ribosomal subunit complex, and this complex migrates along the mRNA until it reaches the start codon (AUG). Next, the UAC anticodon of the initiator Met-tRNA^Met base-pairs with the AUG codon of the mRNA. The large ribosomal subunit then joins the complex, forming the initiation complex (Fig. 3.16).

The elongation and termination stages in pro- and eukaryotes are largely similar. Elongation involves The formation of peptide bonds between adjacent amino acids, with The sequence of incorporated amino acids dictated by the codon order in the mRNA (Fig. 3.17). Let us examine this process in greater detail. Once the initiation complex is formed, the mRNA codon immediately following the AUG codon pairs with the complementary anticodon of the appropriate tRNA, thereby determining which charged tRNA binds to the ribosome (uncharged tRNAs do not bind to ribosomes). If the second triplet in the mRNA is CUG, a leucine-bearing tRNA with the 3'-GAC-5' anticodon joins the ribosomal complex next. Once this tRNA is positioned, a peptide bond is formed between the carboxyl group of methionine and the amino group of leucine, driven by the enzymatic activity inherent to the large subunit. Leucine remains attached to its tRNA while methionine is cleaved from the initiator tRNA, which then dissociates from the ribosome. The resulting methionine-leucine-tRNA^Leu-mRNA complex is translocated through the ribosome, allowing the next mRNA codon to bind to the charged tRNA bearing the corresponding anticodon. If the third mRNA codon is UUU, the next amino acid added to the growing polypeptide chain will be phenylalanine, delivered to the ribosome by a tRNA with the AAA anticodon. When this tRNA is in place, a peptide bond forms between the carboxyl group of leucine and the amino group of phenylalanine. tRNA^Leu then detaches from the ribosome, the peptidyl-tRNA^Phe complex (the tRNA carrying the attached growing polypeptide chain) undergoes translocation, and the subsequent mRNA codon becomes exposed to bind with the appropriate charged tRNA anticodon. These events—binding of a charged tRNA to mRNA via codon-anticodon base-pairing, peptide bond formation, release of the "discharged" tRNA, and translocation—continue until all amino acids encoded by the mRNA have been linked together. Translation proceeds in the 5' to 3' direction at a rate of roughly 15 amino acids per second. As the 5'-end of the mRNA leaves the ribosomal complex, it can bind to another identical complex, enabling a single mRNA molecule to be translated simultaneously by multiple ribosomes.

Fig. 3.16. Translation initiation in a Eukaryotic Cell. The small ribosomal subunit binds to the methionine-charged initiator tRNA (Met-tRNA^Met), and the complex moves along the mRNA until the UAC anticodon of the initiator tRNA base-pairs with the AUG start codon of the mRNA. Subsequently, the large subunit joins the mRNA-tRNA-small subunit complex to establish the initiation complex.

Fig. 3.17. Elongation of the polypeptide chain. A. The second codon (CUG) in the mRNA binds to the anticodon (GAC) of Leu-tRNA^Leu. B. Methionine forms a peptide bond with the leucine delivered by tRNA^Leu, and the now-deacylated initiator tRNA dissociates. C. Translocation of the peptidyl-tRNA-mRNA complex exposes the next codon (UUU). D. The third codon (UUU) pairs with the AAA anticodon of Phe-tRNA^Phe. E. Leucine forms a peptide bond with the phenylalanine delivered by tRNA^Phe, and the deacylated tRNA^Leu dissociates from the ribosome. F. Translocation of the peptidyl-tRNA-mRNA complex exposes the subsequent codon, and so forth.

Elongation continues until the ribosome reaches a UAA, UAG, or UGA codon (a stop or termination codon) (Fig. 3.18). Normally, cells lack tRNAs with anticodons complementary to termination signals. Instead, these signals are recognized by protein release (termination) factors. Upon binding of a release factor to the ribosome, the bond between the final tRNA and the polypeptide is hydrolyzed, releasing the free tRNA, the polypeptide chain, and the mRNA from the ribosome. The ribosome then dissociates into its constituent subunits, which can participate in new rounds of translation.

Following translation, many Polypeptides undergo various post-translational modifications. In most cases, the N-terminal methionine is cleaved off, making the second amino acid the new N-terminal residue. In eukaryotes, certain Proteins undergo Processing, wherein the polypeptide chain is cleaved at specific sites to yield shorter protein molecules with specialized Functions. In some instances, particularly in Eukaryotic cells, phosphate groups, Lipids, CARBOHYDRATES, or other low-molecular-weight compounds are enzymatically attached to specific amino acids. These chemical modifications yield functional proteins tailored for specific cellular roles. The genetic dictionary comprises 64 codons: three function as stop codons, while one (AUG) serves as the start codon (Table 3.2), which also encodes methionine. When an AUG codon appears within the interior of an mRNA molecule rather than at its 5'-end, it is recognized by a different tRNA (Met-tRNA^Met) carrying an unmodified methionine. The amino acid Tryptophan is encoded by a single codon (UGG), whereas all other Proteinogenic Amino Acids are specified by at least two, frequently four, and occasionally six codons. For instance, leucine is specified by six codons: UUA, UUG, CUU, CUC, CUA, and CUG. Synonymous codons are utilized with varying frequencies across different organisms. Among the four Glycine codons, GGA is used in human structural genes in 26% of cases, compared to 9% in Escherichia coli. A similar pattern is observed for stop codons: in humans, the usage frequencies for the UAA, UAG, and UGA codons are 0.22, 0.17, and 0.61, respectively, whereas in E. coli they are 0.62, 0.09, and 0.30. Notwithstanding these variations, METABOLISM/28.html">The Genetic Code remains universal across virtually All living organisms, with only minor exceptions.

Table 3.2. The genetic code and codon usage frequencies in the E. coli and human genomes