S Subunit

(3) SUBUNITS ASSOCIATION

(4) GTP HYDROLYSIS
(5) IF2 RELEASE

Figure 15.6. Sequence of events during initiation of translation in Prokaryotes.

IF2, GDP

Figure 15.6. Sequence of events during initiation of translation in Prokaryotes.

15.3. Initiation in Eukaryotes 15.3.1.Characteristics of Eukaryotic mRNA

The eukaryotic mRNA is synthesized, as pre-mRNA, in the nucleus. From the beginning it is complexed with a large amount of protein. Nuclear pre-mRNA-protein complexes are processed into mRNA-protein complexes (mRNPs) which go through the nuclear membrane. During the transport from the nucleus to the cytoplasm the mRNPs strongly change their protein composition. The protein-to-mRNA weight ratio is about 4:1 in the cytoplasmic mRNPs which are not yet engaged in translation. After initiation of translation and during elongation the mRNA becomes significantly populated by translating ribosomes and looses part of the proteins. Nevertheless, the mRNA within translating polyribosomes is still found to retain a large proportion of protein, the protein-to-mRNA weight ratio being of 2:1 or higher. Thus all eukaryotic mRNA at every stage of its life history exists in the form of mRNP (for reviews, see Preobrazhensky & Spirin, 1978; Spirin, 1996). This seems to be the most characteristic feature of the eukaryotic mRNA.

It follows that the initiation machinery in Eukaryotes deals with highly protein-loaded mRNPs, not with just mRNA. At least three consequences of this fact may be relevant to the initiation process. (1) Secondary and tertiary structures of mRNA may be significantly modified by proteins within mRNPs: on one hand, some "core" proteins are known to melt the structures of mRNA; on the other hand, local hairpins and folds can be stabilized by interacting with proteins. (2) A direct competition between the proteins of mRNPs and the mRNA-binding initiation factors is possible. (3) The structural organization (quaternary structure) of mRNP particles may modulate the accessibility of mRNA for the participants of the initiation process, such as ribosomes and initiation factors.

As mRNA can exist in eukaryotic cytoplasm in two forms, untranslated free mRNP particles and translating polyribosomes, the initiation process may be very different whether ribosomal particles and initiation factors interact with the mRNP and open a "virginal" mRNA for translation, or they bind to a translated mRNA in line after previously initiated ribosomes. In special cases reinitiation after termination, without dissociation of ribosomes from mRNA, is possible when a short open reading frame with termination codon is present upstream of a principal coding sequence (see Section 17.4). Some observations are compatible with the idea that reinitiation can occur also during routine translation of monocistronic messages in circular polyribosomes: if the termination region of mRNA neighbors upon the initiation region the ribosome after termination may reinitiate translation of the same mRNA (see below, Section 15.3.7).

Other characteristic features typical of most, but not all, eukaryotic mRNAs should be also mentioned (for reviews, see Bag, 1991; Merrick & Hershey, 1996). First, eukaryotic mRNAs are mostly monocistronic. Second, they are capped at the 5'-end, with few exceptions. The best known exception is genomic RNA of picornaviruses. Third, many eukaryotic mRNAs are characterized by long 3'-untranslated regions (3'-UTRs) often comparable in length with their coding sequences. At last, the majority of mRNAs in the eukaryotic cytoplasm are polyadenylated at the 3'-end. All these features are also directly relevant to the mechanism of initiation and/or its regulation.

In accordance with the 5'-terminal, cap-dependent initiation as a predominant mode of translation initiation in Eukaryotes, the eukaryotic mRNAs do not possess the Shine-Dalgarno sequence upstream of their initiation codons. As a general rule, no marked complementarity between the 3'-end of the RNA of the small ribosomal subunit and the pre-initiation sequence of mRNA is observed, in contrast to the situation in prokaryotic organisms. It is remarkable that the 3'-terminal 50-nucleotide-long sequences of the ribosomal RNAs of the small ribosomal subunits, including both the 16S RNA of Prokaryotes and the 18S RNA of Eukaryotes, are very conservative in evolution and show high homology, forming a similar stem-loop structure; however, the polypyrimidine CCUCC block of the prokaryotic 16S RNA (the so-called anti-Shine-Dalgarno sequence) is absent from the eukaryotic 18S RNA (Fig. 15.7). This leads to the assumption that, for important reasons, Eukaryotes had to dispose of the complementary recognition between the ribosomal RNA and mRNA in the initial association of ribosomes with the message and develop another way of recognizing the initiation sequence. The new way appears to include the initial association with the 5'-end of mRNA. (This reasoning may be turned upside down: recognizing the 5'-end

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