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Figure 15.1. Schematic representation of two modes of initiation of mRNA translation.

A: Terminal initiation: ribosomal particles (small subunits) bind mRNA at the 5'-end, scan the 5'-untranslated region (5'-UTR) and start translation at the initiation codon (usually AUG); the start is accompanied by association with the large ribosomal subunit.

B: Internal initiation: ribosomal particles (small subunits) interact directly with the ribosome-binding site, including initiation codon, at a distance from the 5'-end of mRNA, associate with large ribosomal subunits and start translation from this point.

15.1.3.Components of Initiation

In all cases the process of initiation of translation involves: (a) small ribosomal subunit (30S or 40S in Prokaryotes and Eukaryotes, respectively); (b) mRNA with its ribosome-binding site (RBS) and initiation codon within; (c) initiator aminoacyl-tRNA; (d) a group of proteins called initiation factors (IFs or eIFs in Prokaryotes and Eukaryotes, respectively); and (e) large ribosomal subunit at the final stage of initiation.

Dissociation of ribosomes into subunits is a pre-requisite for initiation of translation. It is the small ribosomal subunit that initiates the initiation process. It has specific affinities for mRNA, initiator aminoacyl-tRNA and protein initiation factors. It organizes all these components on itself prior to association with the large ribosomal subunit.

Though the small ribosomal subunit has an affinity to any polynucleotide sequence, there are some structural elements in mRNAs which bind the ribosomal particle with an increased strength. Thus such elements, called ribosome binding sites (RBS), are capable of selectively attracting the small ribosomal subunits for initiation. These elements include oligopurinic sequences of Shine-Dalgarno in prokaryotic mRNAs, cap structure with adjacent sequence in eukaryotic mRNAs, special three-dimensional IRES structures in RNAs of picornaviruses, and some other less characterized sequences and structures within prokaryotic and eukaryotic messages. The codon of initiation is usually located on 3'-side of RBS immediately or several to several dozens nucleotides downstream. The predominant initiation codon in Prokaryotes and Eukaryotes is AUG. Related triplets, such as GUG, UUG, AUA, AUU, ACG, can also function as initiation codons in some cases, especially in Prokaryotes, provided RBS is strong.

Initiator aminoacyl-tRNA is always a special methionyl-tRNA (Met-tRNA or Met-tRNA) which cannot participate in elongation and does not interact with EF1A (EF-Tu) or eEF1A. In Prokaryotes it is formylated, thus being formylmethionyl-tRNA (F-Met-tRNA). Thus, methionine is universal starting amino acid residue of all polypeptides and proteins synthesized by ribosomes (later it can be split off by aminopeptidases). Anticodon of the initiator aminoacyl-tRNA is CAU being fully complementary to the main initiation codon AUG, and partly complementary to the rare initiation codons. The most important properties of the initiator aminoacyl-tRNA are that it is not recognized by elongation factor EF-Tu or eEF1A and has a preferable affinity for the P site of the ribosome, rather than to the A site. Prior to the interaction with the ribosome it forms a ternary complex with initiation factor 2 (IF2 or eIF2) and GTP; the complex is the form in which the initiator aminoacyl-tRNA binds with the small ribosomal subunit in the process of initiation.

In addition to IF2 (or eIF2), the small ribosomal subunit cooperates and directly interacts with two more initiation factors: IF1 and IF3, or eIF1 and eIF3, in Prokaryotes or Eukaryotes, respectively. IF3 (eIF3) prevents the association of the small subunit with the large subunit and thus maintains the dissociated state which is competent for initiation. There are all grounds to believe that all the three initiation factors in vivo may be complexed with the small ribosomal subunit prior to its interactions with initiator aminoacyl-tRNA and mRNA; such a complex can be isolated from the cell and is designated as a native 30S or 40S subunit, in contrast to the so-called derived subunit produced by in vitro dissociation of the whole ribosome. The role of the initiation factors sitting on the ribosomal subunit seems to enhance the discrimination of the initiator aminoacyl-tRNA from elongator aminoacyl-tRNAs, and RBS from other mRNA structures. The increased discrimination may be achieved not only by the involvement of the factors in specific positive interactions with the initiator aminoacyl-tRNA and with RBS, but also by decreasing the affinities of the ribosomal subunit for other aminoacyl-tRNAs and, possibly, for nonspecific mRNA sequences.

In Eukaryotes, in addition to the above-mentioned ribosome-binding initiation factors, a group of mRNA-binding initiation factors exists. This group includes, first of all, the factors designated as eIF4A, eIF4B, eIF4E and eIF4F. They have no analogs in Prokaryotes and are engaged in preparing the cytoplasmic mRNA (its region upstream from the coding sequence) for initiation.

15.1.4.Steps of Initiation

As mentioned above, the dissociation of ribosomes into the two subunits is a prerequisite for initiation of translation. The dissociation of the ribosome and the concomitant attachment of the proper initiation factors to the small ribosomal subunit, with the formation of the "native" 30S or 40S particle, can be considered as the first step of initiation Fig. 15.2). Thus, the "native" small ribosomal subunit with IF3 (in the case of the prokaryotic 30S subunit) or eIF3 (in the case of the eukaryotic 40S subunit) starts the initiation process.

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