BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 6. AMINO ACID METABOLISM AND FUNCTIONS. PROTEIN BIOSYNTHESIS

6.10. Protein biosynthesis

6.10.6. Polypeptide chain elongation

During elongation, the incorporation of activated Amino Acids in both eukaryotes and prokaryotes proceeds in three consecutive stages:

✵ recognition and binding of aminoacyl-tRNA to the ribosomal A-site;

✵ peptide bond formation mediated by the peptidyl transferase center of the ribosome;

✵ movement (translocation) of the ribosome by one codon along the mRNA toward its 3'-end.

The translational process proceeds at a relatively low speed compared to METABOLISM/36.html">DNA Replication. Protein Synthesis in prokaryotes occurs at a rate of approximately 20 amino acid residues added per second, whereas DNA replicons synthesize DNA at a rate of about 1000 NUCLEOTIDES per second. This slower pace of Protein Biosynthesis is due to the necessity of accurately positioning the correct aminoacyl-tRNA into the ribosomal A-site and the potential for proofreading and correction mechanisms during this process.

Elongation in prokaryotes. The First stage of the elongation cycle involves the precise placement of the appropriate aminoacyl-tRNA into the ribosomal A-site. In Bacteria, this process is catalyzed by the EF-Tu protein factor, a monomer (molecular mass 47·10³) containing a GTP-binding center (Elongation Factor Tu). *E. coli* contains 135,000 molecules of EF-Tu, highlighting its high Abundance within The Cell. The aminoacyl-tRNA–EF-Tu–GTP ternary complex (nearly all aminoacyl-tRNA molecules *in vivo* exist as part of such complexes) can freely diffuse to the ribosomal A-site. Upon correct base-pairing—when the aminoacyl-tRNA anticodon is complementary to the mRNA codon in the A-site—the complex interacts productively with the ribosome. The recruitment of aminoacyl-tRNA to the ribosome is driven by the Hydrolysis of GTP to GDP and Pi, followed by Conformational Changes in the EF-Tu–GDP complex and its release from the ribosome. Regeneration of the active EF-Tu–GTP complex is facilitated by the protein factor EF-T_s (molecular mass 35·10³) and GTP (Fig. 6.41). Consequently, the aminoacyl-tRNA becomes properly positioned in the ribosomal A-site, primed for the next stage: peptide bond formation.

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Fig. 6.41. First stage of the elongation cycle in prokaryotes: codon recognition and binding of aminoacyl-tRNA to the ribosomal A-site

The Second Stage of the elongation cycle is The formation of a peptide bond via a transpeptidation reaction. A nucleophilic attack by the nitrogen atom of the α-amino group of the aminoacyl-tRNA (A-site) on the carbonyl carbon of the peptidyl-tRNA (P-site) results in The transfer of the formylmethionine residue to the NH₂ group of the aminoacyl-tRNA, thus forming the first peptide bond in the nascent protein molecule (Figs. 6.42; 6.43). Catalysis of this reaction within the peptidyl transferase center of the ribosome is mediated by the 23S rRNA of the large subunit, which exhibits catalytic activity and belongs to the class of ribozymes.

Fig. 6.42. Second stage of the elongation cycle in prokaryotes: peptide bond formation

At this stage, hybrid sites are formed on the ribosome: the anticodons remain in the A- and P-sites respectively, the 3'- and 5'-ends of the dipeptidyl-tRNA shift into the P-site of the ribosome, while the deacylated initiator tRNA positions its 3'- and 5'-ends in the ribosomal E-site (Fig. 6.44).

During the Third Stage—translocation, catalyzed by the protein factor EF-G (molecular mass 80·10³) coupled with GTP hydrolysis—the peptidyl-tRNA is fully shifted (along with its anticodon and the corresponding mRNA codon) into the ribosomal P-site. This precise movement of the ribosome relative to the mRNA (from the 5'- to the 3'-end) by exactly one nucleotide triplet (codon) promotes the transition of the deacylated tRNA into the ribosomal E-site for subsequent release into the Cytosol, while placing the next mRNA codon into the A-site to dictate The addition of the next amino acid to the growing peptide chain (Fig. 6.45).

Fig. 6.43. Scheme of peptide bond formation:

P-site containing peptidyl-tRNA (I);

A-site containing aminoacyl-tRNA (II)

Fig. 6.44. Hybrid ribosomal sites

Fig. 6.45. Third stage of the elongation cycle in prokaryotes: translocation

Thus, during the elongation stage, the polypeptide chain is sequentially extended by one amino acid residue in strict accordance with the codons in the mRNA molecule.

Elongation in eukaryotes. The elongation process in eukaryotes does not differ fundamentally from the prokaryotic microcycle, although it features a higher level of Organization and compartmentalization of components. Protein factors eEF-1A (molecular weight 50·10³) and eEF-2 (molecular weight 100·10³) are functional analogs of EF-Tu and EF-G in prokaryotes. The regeneration of eEF-1A·GDP to eEF-1A·GTP takes place with the participation of eEF-1B. Peptide bond formation is catalyzed by the large 60S subunit, specifically its 28S rRNA. To date, a group of RNA molecules with enzymatic properties (ribozymes) has been discovered. It is believed that ribozymes are "relics" of an early period of evolution.

As a result of elongation, the peptide chain is extended by one amino acid residue. The repetition of cycles corresponding to the number of sense codons in the mRNA completes the entire elongation phase.