Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Protein synthesis on ribosomes
Translation elongation

Thus, the eukaryotic complex (80S-ribosome-Met-tRNA_i^Met) assembled at the start codon is ready for the stepwise elongation of the protein chain. Similar to the Translation initiation stage, a set of specialized Proteins—elongation factors (EF)—is required.

The MAIN STAGES OF elongation are:

1) binding of the next aminoacyl-tRNA,

2) conformational change of the ribosome,

3) Formation of the peptide bond,

4) shift, or translocation, of the ribosome by one codon along the mRNA.

At the completion of translation initiation, Met-tRNA_i^Met is bound to the P-site of the 80S ribosome (Figure 43). This site is designated as "P" because it is precisely where the tRNA chemically linked to the growing polypeptide chain resides.

The second aminoacyl-tRNA is delivered to the ribosome as a ternary complex in combination with EF1a and GTP and binds to the A-site, so named because it accepts the aminoacylated tRNA (Figure 43, step 1). If the anticodon of the second aminoacyl-tRNA correctly pairs with the second codon of the mRNA, the GTP bound to EF1a undergoes Hydrolysis.

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Figure 43 - Stages of translation elongation

GTP hydrolysis stimulates a conformational change in the ribosome (in Figure 43, the initially "oval" ribosome becomes "square"). This conformational change of the ribosome

✵ firmly anchors the aminoacyl-tRNA in the A-site,

✵ induces the release of the dephosphorylated complex (EF1a-GDP),

✵ brings the 3'-end of the tRNA at the A-site into close proximity with the 3'-end of Met-tRNA_i^Met in the P-site (Figure 43, step 2).

GTP hydrolysis, and consequently tight binding to the ribosome, does not occur if the aminoacyl-tRNA anticodon fails to pair with the codon in the A-site. In this case, the ternary complex diffuses away, leaving the A-site vacant for subsequent complexes (aminoacyl-tRNA-EF1a-GTP) to attempt binding until a correct codon-anticodon combination is formed and pairing takes place. This phenomenon ensures the "assembly fidelity" of the polypeptide chain.

A three-dimensional model of the ribosome is shown in Figure 44.

Figure 44 - Model of the 70S E. coli ribosome with three tRNAs in the E, P, and A sites

When Met-tRNA_i^Met is located in the P-site and the second aminoacyl-tRNA is firmly bound in the A-site, the α-amino group of the second amino acid reacts with the initiator tRNA (Met-tRNA_i^Met), which is activated by Methionine, thereby forming a peptide bond (Figure 43, step 3, Figures 32 and 35). This peptidyl transferase reaction is catalyzed by the large rRNA, which spatially orients the interacting atoms with exquisite precision to facilitate the reaction.

The catalytic activity of the large rRNA itself was proven by the fact that it catalyzed the peptidyl transferase reaction even under conditions where all small proteins were removed from the large ribosomal subunit. Another argument supporting the catalytic activity of the large rRNA comes from crystallographic studies of the ribosomal Spatial Structure, which revealed that no ribosomal proteins are present near the site where peptide bond synthesis occurs.

Following peptide bond synthesis, the ribosome shifts (a process known as translocation) along the mRNA by one codon. This translocation step is driven by the GTP hydrolysis of the eukaryotic complex (EF2-GTP). As a result of translocation, tRNA_i^Met, now lacking the activated methionine, shifts to the E-site—the exit site for tRNA release from the ribosome. Meanwhile, the second tRNA, now covalently linked to the dipeptide, moves into the P-site (Figure 43, step 4).

Thus, translocation returns the ribosome to its "initial" conformation, a state in which the A-site is vacant and capable of accepting the next aminoacylated tRNA in complex with EF1a and GTP, initiating the second (and subsequently all subsequent) elongation cycle (Figure 43, step 5).

These elongation cycles, illustrated in Figure 43, add one amino acid per cycle to the C-terminus of the growing polypeptide chain in accordance with the mRNA codon sequence, repeating until a stop codon is reached.

Furthermore, during step 2 of each cycle, the conformational change experienced by the ribosome triggers the release of the deacetylated tRNA from the E-site. As the growing polypeptide chain elongates, it passes through a specialized tunnel within the body of the large ribosomal subunit and emerges on the opposite side, directly across from the interface where the large subunit interacts with the small subunit (Figure 44).

The positioning of tRNAs in the A, P, and E sites has also been visualized through the Determination of the bacterial ribosome crystal structure. This is illustrated in Figure 45 to the extent possible in a black-and-white rendering.

Figure 45 — Model of the 70S ribosome from N. thermophilus

It was found that base pairing occurs between the tRNAs located in the A and P sites (Figure 45, top right).

Such an RNA-RNA hybrid, held together by only three nucleotide pairs, is unstable under physiological conditions. However, multiple interactions between rRNA molecules on the one hand and the core domains of tRNA (such as the D- and TΨC loops (Figure 36)) on the other, stabilize the tRNAs in the A and P sites, while other RNA-RNA interactions "finely" monitor the accuracy of codon-anticodon pairing, ensuring The fidelity of METABOLISM/28.html">The Genetic Code reading.