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Figure 15.15. tRNA-like structure and pseudoknots at the 3'-end of tobacco mosaic virus (TMV) RNA. A: The predicted secondary structure and its L-arrangement. B:. The model of three-dimensional folding of the same region .

(Reproduced from K. Rietveld, K. Lenschooten, C.W.A.Pleij & L. Bosch, EMBO J. 3, 2613-2619, 1984, with permission).

same time the leader sequence of the TMV RNA, i.e. the 5'-UTR element, strongly competed for the binding with this protein and was found to be even a more efficient competitor than the homologous 3'-UTR sequence. All this suggests that (1) both elements, 3'-UTR pseudoknot domain and 5'-UTR leader domain, are specifically recognized by the same protein factor present in plant cell extract, and (2) the interaction of the binding protein with the 5'-UTR domain is tighter than that with the 3'-UTR domain. One may speculate that the binding protein is one of the ubiquitous RNA-binding initiation factors which normally interacts with 5'-UTRs of cellular mRNAs, but also recognizes the viral 3'-UTR element.

A potent translational enhancer domain of a different type has been shown in the 3'-UTR of satellite tobacco necrosis virus (STNV) RNA. This is a monocistronic messenger that lacks both the cap structure and the poly(A) tail. A bulged hairpin structure responsible for the enhancing effect is located immediately downstream from the coding sequence (Danthinne et al., 1993; Timmer et al., 1993). The proper structure of the short 5'-UTR is required for the enhancing effect of the 3'-UTR domain. Protein cofactors or initiation factors that could mediate the effect are not known.

Thus, like in the case of the enhancer function of the poly(A) tail, it is tempting to hypothesize that the effect of viral RNA enhancers is achieved through a local, protein-mediated contact of the 3'-UTR structure with the initiation regions of mRNA. The alternative may be the formation of a protein complex at the 3'-portion of mRNP which fixes the global mRNP structure in a conformation better available for translational initiation (global structural effect).

The enhancing effect of 3'-UTR sequences of cellular mRNAs can also be suspected in some cases. For example, it was reported that the removal of a major portion of the ornithine decarboxylase (ODC) mRNA 3'-UTR is inhibitory for translation (Manzella & Blackshear, 1990), and that the insertion of the ODC 3'-UTR downstream of the termination codon of a reporter mRNA partially relieves the suppression of translation imposed by the ODC 5'-UTR (Grens & Scheffler, 1990). There are no indications concerning proteins which could mediate these effects.

15.3.8.Sequence of Events

Step 1. In order to enter the initiation process the ribosome must be dissociated into the subunits. It is believed that the decisive role in the dissociation and the prevention of reassociation is played by IF1A and eIF3 that interact with the 40S subunit. eIF1 and eIF2 may also join the complex called native 40S ribosomal particle. The 60S subunit interacts with eIF3A (eIF6) and some other proteins forming the native 60S ribosomal particle.

Step 2. The "classical" scenario is the activation of eIF2 with GTP and the subsequent binding of the initiator Met-tRNAi to the GTP-form of eIF2. This event is usually considered to occur as the interaction of the three free components, with the formation of the ternary complex Met-tRNAi:eIF2:GTP in solution:

eIF2 + GTP + Met-tRNAi----»—eIF2:GTP + Met-tRNAi---->■ • • •Met-tRNAi:eIF2:GTP.

Then the ternary complex attaches to the native 40S particle:

40S :eIF3:eIF 1A + Met-tRNAi:eIF3:GTP----•40S :eIF3:eIF 1A:eIF2:GTP :Met-tRN Ai.

However, since eIF2 is likely to be attached already to the native 40S particle, the formation of its complex with GTP and Met-tRNA may take place rather on the particle than in solution (Fig. 15.16):

-• • 40S:eIF3:eIF 1A:eIF2:GTP + Met-tRNAi---->-

The ribosomal complex formed is often designated as 43S initiation complex.

Step 3. The next step is usually considered to be the binding of the 43S initiation complex to mRNA and thus the formation of the so-called 48Sinitiation complex (see Fig. 15.13). The mRNA binding proceeds with the participation of eIF4E, eIF4F, eIF4A and eIF4B. This step includes the primary association of the ribosomal particle with mRNA (with cap-structure or IRES), the movement of the ribosomal particle along 5'-UTR downstream (scanning), and the selection of the initiation codon. The "classical" model presumes that the cap-binding factors eIF4E and eIF4F interact with the cap-structure of mRNA, and then eIF4A and eIF4B come and perform ATP-dependent unwinding of the 5'-UTR, this being a prerequisite for landing the 43S initiation complex on mRNA and subsequent scanning.

A more likely model based on information available about the intracellular localization of initiation factors and their complex formation capabilities suggests that the cap-binding complex (eIF4F) and the helicase complex (eIF4A:eIF4B) are assembled on the ribosomal particle in the presence of mRNA. Therefore, according to this model, it is the ribosomal particle with the initiation factors that binds to the cap structure and then successively unwinds the downstream sequence thus moving along mRNA and scanning the sequence (Fig. 15.16). The principal role in assembly of the cap-binding and helicase complexes on the native 40S ribosomal particle may belong to eIF3 which is capable of interacting with eIF4F (with eIF4G subunit) and eIF4B. It is possible that eIF4E is pre-bound to the cap-structure of mRNA competent for translation, whereas eIF4G subunit of eIF4F and eIF4B are associated with the native 40S ribosomal particle. In such a case the complete cap-binding complex (eIF4F) is assembled upon binding of the native ribosomal particle with the cap-structure, followed by the assembly of the helicase complex (eIF4F:eIF4B or eIF4A:eIF4B) on the particle.

It cannot be excluded that this step (mRNA binding) precedes the step of the initiator Met-tRNAi binding. In other words, Met-tRNAi binding may proceed after the native ribosomal particle with full set of initiation factors including eIF2 associates with mRNA, scans its 5'-UTR and finds the initiation region. The eIF2:GTP-dependent binding of the Met-tRNAi may be significantly stimulated by the presence of the

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