BIOCHEMISTRY - Textbook - L. I. Ostapchenko - 2012

Chapter 6. AMINO ACID METABOLISM AND FUNCTIONS. PROTEIN BIOSYNTHESIS

6.10. Protein biosynthesis

6.10.5. Translation Initiation. Specific Features of the Initiation Process in Prokaryotes and Eukaryotes

The first ribosomal stage of template polypeptide synthesis is Translation initiation. During Protein Synthesis, mRNA decoding proceeds in the 5'- to 3'-direction, ensuring Peptide Synthesis from the N- to the C-terminus of the molecule, which was proven in 1961 by H. Dintzis.

The fundamental function of translation initiation is the error-free Location OF THE initiation codon by the ribosome, which determines the start and reading frame of the mRNA. The second function is the delivery of the donor substrate into the P-site of the ribosome, since for the next elementary elongation act—the extension of the polypeptide chain—the peptidyl center must be involved in the translation process. The third important aspect is the crucial role of the initiation mechanism in the Regulation of Protein Biosynthesis at the translational level. Initiation itself is the primary regulatory stage of METABOLISM/35.html">Protein Biosynthesis, determining the translation intensity of various mRNAs, its arrest in response to various intracellular signals, or the induction of translation of "silent" mRNAs. Moreover, varying translation rates across different mRNAs determine the required ratios of protein production in the Organism. To perform these Functions, 30(40)S + 50(60)S → 70(80)S Ribosomes, initiator aminoacyl-tRNA, initiation codons within the mRNA, protein initiation factors, and GTP are required. The processes of translation initiation in pro- and eukaryotes are generally similar, although the functional complexity of template synthesis in eukaryotes affects the operation of their translational apparatus.

Initiation in prokaryotes. Translation of natural mRNAs begins with AUG or GUG codons. As a rule, the initiation codon is most frequently AUG, and the first amino acid at the N-terminus of the polypeptide is Methionine. All organisms possess two different Met-tRNA^Met species, one of which is the initiator, while the second accepts methionine residues and incorporates them during the elongation stage. Methionyl-tRNA synthetase is the same in both cases. The methionine incorporated during the initiation stage in prokaryotes is formylated (Fig. 6.37, 1).

The process of formylmethionyl-tRNA^fMet formation (Fig. 6.37, 2) proceeds in two stages. First, mediated by the corresponding aminoacyl-tRNA synthetase, methionine is attached to tRNA^fMet. At the next stage, transformylase transfers the formyl group from N¹⁰-formyltetrahydrofolic acid to the amino group of methionine, forming fMet-tRNA^fMet. Transformylase is a more specific enzyme than methionyl-tRNA synthetase, as it catalyzes the process exclusively with tRNA^fMet, recognizing specific Structural Features of its molecule. The peculiarities of the primary, secondary, and even Tertiary Structure of initiator aminoacyl-tRNAs lead to their interaction with protein initiation factors, facilitating their subsequent placement exclusively in the P-site of the ribosome. Thus, the formylation process has profound chemical and biological significance: by blocking the NH₂ group of methionine, Protein synthesis proceeds in the NH₂ → COOH direction, and formylmethionyl-tRNA^fMet helps mRNA locate the specific site on the 30S subunit that ensures the translation of information via the sequential Incorporation of Amino acid residues into the protein molecule. During protein Processing, the formyl group may be removed by the action of deformylase, while the methionine residue is cleaved in some cases with the participation of methionyl aminopeptidase.

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Fig. 6.37. Scheme of N-formylmethionyl-tRNA synthesis in prokaryotes

Studies by J. Shine and L. Dalgarno established that the 5'-AUG start codon is preceded by a universal structure—a polypurine sequence (3–9 NUCLEOTIDES, e.g., AAGGAG)—which is characteristic of the ribosome-binding sites of prokaryotic mRNAs and is complementary to a polypyrimidine sequence located at the 3'-end of the 16S rRNA molecule. This structure was named the Shine-Dalgarno sequence (Fig. 6.38). Thus, Watson-Crick interactions stabilize THE POSITION OF mRNA within the binding center of the 30S subunit of the prokaryotic ribosome (Fig. 6.39).

Fig. 6.38. Stabilization of the prokaryotic initiation complex

Interaction of mRNA (Shine-Dalgarno sequence) with 16S rRNA (polypyrimidine sequence)

Fig. 6.39. Initiation of Protein synthesis in prokaryotes

CAV – codon-anticodon interaction

Translation initiation in prokaryotes involves three protein factors, namely: IF-1, IF-2, and IF-3 (Initiation Factors), with molecular weights of 9·10³, 95·10³, and 22·10³, respectively. IF-1 participates in the dissociation of ribosomal subunits and the stabilization of 30S subunit interactions with other protein factors and fMet-tRNA^fMet, while preventing the binding of aminoacyl-tRNA to the ribosomal A-site. IF-3 prevents premature association of the 30S with the 50S subunit and facilitates the recruitment of IF-2, as well as the correct positioning of fMet-tRNA^fMet and the initiation codon in the ribosomal P-site. IF-2 is the major protein initiation factor; it exhibits a high affinity for GTP, forming complexes with it, interacts with fMet-tRNA^fMet and the 30S ribosomal subunit, and is considered the stabilization factor for the entire initiation complex.

Formation of the prokaryotic 70S initiation complex. At The First stage, mediated by IF-1, IF-3 attaches to the 30S subunit. Subsequently, the complementary interaction of the Shine-Dalgarno sequence in the mRNA with the corresponding regions in the 16S rRNA facilitates the positioning of the AUG start codon in the ribosomal P-site. At the next stage, the fMet-tRNA^fMet·IF-2·GTP complex joins the complex consisting of IF-1, IF-3, mRNA, and the 30S subunit, leading to codon-anticodon interaction in the ribosomal P-site. At the Third Stage, the 50S subunit joins the complex, which results in the Hydrolysis of GTP to GDP and orthophosphate due to the GTPase activity of IF-2, the release of protein initiation factors, and The formation of the prokaryotic 70S initiation complex. The energy released during GTP hydrolysis is directed toward stabilizing the 70S initiation complex. The ribosome, with a filled P-site and a vacant A-site, is primed to enter the elongation process (Fig. 6.39).

Prokaryotes are characterized by internal initiation. In this case, the small ribosomal subunit associates directly with a local mRNA structure containing the initiation codon, independently of the 5'-end of the mRNA and its distance from the beginning of the coding sequence. Since a single long prokaryotic mRNA often contains multiple coding sequences for different Proteins (polycistronic mRNAs), this mechanism ensures the translation initiation of several coding sequences within such an mRNA independently of one another.

Translation initiation in eukaryotes. The initiation process is much more complex in eukaryotes, where the small ribosomal subunit typically first binds to the 5'-end of the mRNA and then moves along the mRNA chain, scanning it until it encounters the initiation codon. The greatest differences in translational processes between pro- and eukaryotes are observed precisely at the initiation stage. Eukaryotic mRNA possesses a complex Secondary structure enriched with self-complementary hairpin-like folded regions, contains modified 5'- and 3'-ends (a 5'-cap and a 3'-poly(A) tail), and forms complexes with RNA-binding proteins (informosomes). The initiator methionyl-tRNA in eukaryotes is not formylated and possesses A number of structural features compared to the elongation Met-tRNA^Met. Eukaryotes feature a significantly larger number of protein initiation factors—eIFs (Eukaryotic Initiation Factors).

Eukaryotic protein initiation factors are divided into two main groups:

✵ those bound to mRNA that prepare it for the initiation process: the heteromultimeric eIF-4F complex (eIF-4A, eIF-4E, eIF-4G) and eIF-4B;

✵ those that interact with ribosomal subunits, mediating their association/dissociation as well as binding to the initiator Met-tRNA_i^Met and mRNA: eIF-1, eIF-2, eIF-3, eIF-5, and eIF-6.

The structural features and Functions of the main eukaryotic protein initiation factors (according to B. Negrutskii) are presented in Table 6.9.

Thus, the preparation of eukaryotic mRNA for initiation occurs in stages: eIF-4E binds to the cap structure at the 5'-end of the mRNA; eIF-4B forms a complex involving eIF-4A, inducing its RNA-dependent helicase activity—unwinding the Introduction/11.html">Secondary structure of the 5'-end of the mRNA using the energy of ATP hydrolysis; eIF-4G coordinates the action of all factors. Simultaneously, the ternary complex—the initiator Met-tRNA_i^Met along with eIF-2·GTP and the 40S subunit, with the participation of factors eIF-1 (1A)—forms the 43S pre-initiation complex, which, in turn, by joining (via eIF-3) the mRNA prepared for initiation, stabilizes the formation of the 48S pre-initiation complex. Thus, placing the 40S subunit onto the 5'-end of the mRNA initiates the ATP-dependent scanning of the non-coding region of the template to locate the start codon; the energy of ATP hydrolysis is spent on overcoming helical regions in the untranslated region of the mRNA. Upon finding THE START OF the coding sequence, codon-anticodon complementary interaction takes place between AUG and the anticodon within Met-tRNA_i^Met, and conformational rearrangements of the complex are observed.

Table 6.9

Eukaryotic Initiation Factors

The subsequent joining of the 60S subunit to form the 80S eukaryotic initiation complex is accompanied by the hydrolysis of GTP bound to eIF-2, mediated by eIF-5 and eIF-2B. Following the dissociation of all initiation factors, an 80S ribosome is formed containing a P-site with the mRNA AUG codon base-paired with the corresponding anticodon of Met-tRNA_i^Met. Thus, an engaged ribosomal P-site alongside an available free A-site indicates that the eukaryotic ribosomal machinery is ready to enter the active elongation phase.

The complexity of the initiation process in eukaryotes is also related to the involvement of the poly(A) tail at the 3'-end of the mRNA in forming the initiation complex through interaction with the 5'-end of the mRNA. This structure is stabilized by poly(A)-binding proteins (PABPs), which have an affinity for initiation factors—specifically the cap-binding proteins eIF-4G and eIF-4E—bringing both ends of the mRNA into close spatial proximity (Fig. 6.40). Such a pseudo-closed conformation of eukaryotic mRNA is efficient for recruiting initiation complexes and regulating Gene Expression.

Fig. 6.40. Formation of the eukaryotic initiation complex:

PABP — poly(A)-binding protein