Initiation Of Translation

15.1. General Principles

The vacant ribosomal particles after termination may be again involved in translation. Several mechanisms of initiation of translation of a new message, or re-initiation of translation of the next coding sequence of the same polycistronic mRNA (in Prokaryotes), or re-initiation of translation of the same monocistronic mRNA (in Eukaryotes) by vacant post-termination ribosomes have been evolved in the living matter.

15.1.1.Significance of Initiation Stage

Message-dependent elongation of peptide performed by the ribosome requires the presence of a peptidyl-tRNA as a substrate to react with a newly arrived, codon-bound aminoacyl-tRNA. In other words, the donor substrate must be present in the ribosomal P site to be elongated by the next amino acid residue. Hence, in order to initiate translation, a special mechanism must exist that inserts a donor substrate into the P site of an empty (non-translating) ribosome. This mechanism of the translation initiation will be discussed below.

The initiation mechanism may be considered as a modified elementary elongation cycle (Section 9.1, Fig. 9.1). A special initiator aminoacyl-tRNA, in the complex with a special initiation factor and GTP, binds to an initiator codon of mRNA at the ribosome. This stage looks like the aminoacyl-tRNA:EF1A:GTP binding stage in the elongation cycle. However, in the process of binding, the initiator aminoacyl-tRNA is directed to the P site of the ribosome, rather than to the A site. This correlates with the fact that the corresponding initiation factor (IF2 in the case of Prokaryotes) is structurally and functionally similar to EF2 (EF-G in Prokaryotes). Thus, elements of translocation can be seen in the process of the initiator aminoacyl-tRNA binding. Then the initiator aminoacyl-tRNA occupying the P site serves as a donor substrate in the subsequent reaction with an A-site-bound elongator aminoacyl-tRNA, i.e. it functionally mimics the peptidyl-tRNA in the elongation cycle. In Prokaryotes the initiator aminoacyl-tRNA resembles a peptidyl-tRNA also chemically: its amino group is formylated, i.e. involved in amide bond.

Initiation of translation matters not simply the beginning of the peptide elongation process. It is also the start of the message read-out at a specific point of a message polynucleotide. Since the start of the coding sequence does not coincide with the 5'-end of the message polynucleotide but is removed from the 5'-end, sometimes by a significant distance, a precise recognition of the first codon is required. Very precise start point is of especial importance in translation because it sets a proper reading frame for all the subsequent coding sequence of a given mRNA, for its triplet-by-triplet readout. Thus the finding of the first codon from which both the start and the frame are counted is another fundamental function of the initiation mechanism.

Furthermore, initiation constitutes the principal step at which protein synthesis is controlled at the translational level (Sections 16 and 17). Regulation at the translational level may be discussed in terms of either permitting or preventing the initiation of the mRNA readout by ribosomes. Selective or preferential translation of certain mRNA species (or mRNA cistrons in Prokaryotes) and translational inactivation of other mRNAs are achieved precisely in this way. In addition, differential rates of initiation with different mRNAs (or different cistrons) determine the ratio of production of corresponding proteins. Generally, the initiation mechanism exerts its intrinsic selectivity towards different messages and also serves as a target for the action of positive and negative regulatory signals.

15.1.2.Prokaryotic and Eukaryotic Modes of Initiation

There are two modes of initiation in the living nature. One requires and starts with the 5'-end of mRNA, though the initiation codon is always positioned at some distance from the end. It is assumed that the ribosome associates with the 5'-end, usually modified ("capped", see Fig. 2.4), and then scans the downstream nucleotide sequence until it encounters the initiation codon (Fig. 15.1 A). This mode of the so-called 5'-terminal initiation is predominantly used by Eukaryotes. The mechanism includes special mRNA-binding protein factors for the recognition of the capped 5'-end, ATP-dependent unwinding of RNA helices during scanning, fixation of the ribosome at the initiation codon, etc. The initiation of this type takes place on monocistronic mRNA pre-synthesized in the nucleus, transported into the cytoplasm and complexed with protein to form mRNP.

The other initiation mode is the so-called internal initiation when the ribosome associates directly with the mRNA structure containing initiation codon within it or nearby, independently of the mRNA end (Fig. 15.1 B). This mode is essential in Prokaryotes. Eukaryotes, however, also use the mode of internal initiation in translation of mRNAs of some special classes. In the typical prokaryotic version of the internal initiation the ribosome can associate with nascent mRNA and start translation during mRNA synthesis (coupled transcription/translation), and the multiple coding sequences (mRNA cistrons) of polycistronic mRNAs can be independently initiated and translated.

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