S S

Figure 15.8. Scanning model for initiation of translation in Eukaryotes (M. Kozak, Cell 15, 1109-1123, 1978; 22, 7-8, 1980; 459^167, 1980): the ribosomal particle (40S subunit) with initiation factors moves phaselessly along mRNA from the 5'-end downstream and unwinds its secondary/tertiary structure using the energy of ATP hydrolysis until it encounters an AUG triplet in the proper context; this AUG serves as the initiation codon for the following translation.

are not in a proper structural environment. Nearly all of the functional initiation AUG codons of eukaryotic mRNAs are preceded by the triplet beginning with the purine nucleotide, in most cases with A. It is assumed that when the initiating 40S particle scans the template, it preferentially recognizes the sequence PuNNAUG (where N can be any nucleotide residue) as the correct initiation site (Kozak, 1981, 1989a). AUG triplets with the preceding PyNN triplet seem to be "weak" initiators and can be skipped without initiation. In addition, the functional initiation codons have G as a preferential neighbor at the 3'-side and the C-rich pentanucleotide sequence at the 5'-side; for example, the sequence CCACCAUGG provides a proper initiating "strength" to the AUG triplet in it.

There are special cases, particularly among some virus-induced mRNAs, where the phenomenon of "two initiation points" is observed: the first AUG triplet is recognized as initiation codon only by a portion of scanning ribosomes, thus being just a "weak" initiator, while the other ribosomes skip it without initiation and initiate at the next "strong" AUG. In such cases, mRNA behaves as functionally bicistronic, since the synthesis of two different polypeptides is initiated and proceeds on the overlapping nucleotide sequences.

As an alternative to the scanning model, another model has been proposed (Sonenberg, 1991). It presumes that the cap structure and the initiation codon region, being far apart along the sequence, are close to each other in the three-dimensional structure formed by the cap with the 5'-UTR. According to this model, the initiating ribosomal particle recognizes not just the cap structure, but a three-dimensional structural element including both the cap and the initiation codon.

It should be added that the eukaryotic initiation mechanism is much more strict as to the nature of the initiation triplet, in comparison with prokaryotic systems: AUG is almost exclusively used as the initiation codon in eukaryotic mRNAs (reviewed by Kozak, 1983). There are very rare cases of starting at other triplets, such as GUG, UUG, CUG and ACG.

15.3.3.Internal Ribosome Entry Site

In addition to the cap-dependent 5'-terminal initiation most commonly used in Eukaryotes, the internal initiation mechanism is found to be also inherent in eukaryotic systems (for reviews, see Meerovitch et al., 1991; Jackson, 1996; Ehrenfeld, 1996). It has been found that the eukaryotic ribosomes (initiating 40S particles) are capable of recognizing some special three-dimensional structural elements inside mRNA molecules, binding to them and starting either scanning or translation from them. These elements are designates as "internal ribosome entry sites", or IRESes. The best known case of internal initiation in eukaryotic systems is that of translation of picornavirus RNAs. Among cellular mRNAs of the eukaryotic cell the products of genes that control growth and differentiation often have long 5'-UTRs with many AUGs, and they are suspected to possess IRESes and use the internal initiation mechanism; for some of such mRNAs the cap-independent internal initiation has been proven. The best studied cases of cellular mRNAs with internal initiation are the Antennapedia mRNA of Drosophila, and the mammalian BiP mRNA which codes for immunoglobulin heavy-chain binding protein, also called 78 kDa glucose-regulated protein (GRP 78) (Macejak & Sarnow, 1991; OH et al., 1992).

The structure of IRES is in no case known yet, although some primary and secondary structural motifs of the picornavirus IRESes have been identified. The picornaviral IRES comprises the sequence of about 450 nucleotide residues long. This segment of RNA appears to form a compact three-dimensional structure having an affinity for the initiating 40S ribosomal particle ("ribosome landing pad"). With respect to the IRES secondary structure, picornavirus RNAs can be divided into two groups with high inter-group conservation, but little similarity between the groups. Thus, enteroviruses (e.g., poliovirus) and rhinoviruses have one type of IRES folding, whereas cardioviruses (e.g., encephalomyocarditis virus) and aphthoviruses (foot-and-mouth disease virus) possess another secondary structure pattern of their IRESes (Fig. 15.9). Despite very unlike secondary and probably tertiary structure both types of IRESes effectively provide internal initiation, that is the selective association with ribosomal particles and subsequent initiation of translation.

The picornaviral IRES is followed by another important element, the so-called starting window (Pilipenko et al., 1994). This is a segment of about 10 nucleotides long located at the fixed distance of 16 or 17 nucleotides from the 3'-boundary of IRES (Fig. 15.10). If an AUG triplet is found within this segment, the ribosomal particle associated with IRES starts either translation, or movement along RNA with searching for the next AUG (scanning). The sequence in this segment is not of great importance for

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