Protein translation eukaryotic initiation factors

Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome. Once the mRNA binds to the ribosome, the latter finds the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (elFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps (1) dissociation of the ribosome into its 40S and 60S subunits (2) binding of a ternary complex consisting of met-tRNAf GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex (3) binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex and (4) combination of the 43S initiation complex with the 60S ribosomal subunit to form the SOS initiation complex. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

Figure 7.5 Model of ferritin (and erythroid a-aminolaevulinate synthase) translation/ribosome binding regulation by IRP. In (a), with IRP not bound to the IRE (1) binding of the 43S preinitiation complex (consisting of the small ribosomal 40S subunit, GTP and Met-tRNAMet) to the mRNA is assisted by initiation factors associated with this complex, as well as additional eukaryotic initiation factors (elFs) that interact with the mRNA to facilitate 43S association. Subsequently (2), the 43S preinitiation complex moves along the 5 -UTR towards the AUG initiator codon, (3) GTP is hydrolysed, initiation factors are released and assembly of the 80S ribosome occurs. Protein synthesis from the open reading frame (ORF) can now proceed. In (b) With IRP bound to the IRE, access of the 43S preinitiation complex to the mRNA is sterically blocked. From Gray and Hentze, 1994, by permission of Oxford University Press.

Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate. The prefix elF denotes a eukaryotic initiation factor. For example, eIF-4E is a protein that binds directly to the 7-inethylguanosine cap (p. 846), whereas eIF-2, in association with GTP, delivers the met-tRNA to the ribosome. The difference in initiation mechanism between prokaryotes and eukaryotes is, in part, a conseciuence of the ence in RNA processing. The 5 end of mRNA is readily available to ribosomes immediately after transcription in prokaryotes. In contrast, pre-mRNA must be processed and transported to the cytoplasm in eukaryotes before translation is initialed. The 5 cap provides an easily recognizable starting point. In addition, the complexity of eukaryotic translation initiation provides another mechan ism for regulation of gene expression that we shall explore further in Chapter 31. 

This is the part of eukaryotic translation that is the most different from that in prokaryotes. Thirteen more initiation factors are given the designation elF, for eukaryotic initiation factor. Many of them are multisubunit proteins. Table 12.4 summarizes pertinent information about these initiation factors. 

TABLE 13.9 Eukaryotic initiation factors for protein translation... 

Maskin is a cytoplasmic polyadenylation element-binding protein-associated factor. Dormant state of maternal mRNAs in immature oocytes is maintained by an abortive interaction of this protein with the eukaryotic initiation factors 4E and 4G. Phosphorylation of maskin promotes the dissociation of this interaction, thereby allows the dormant mRNAs to be translated actively. Aurora phosphorylation of maskin is reported to be involved in protein synthesis in maturing clam and Xenopus oocytes and in centrosome-dep>endent microtubule assembly at mitosis (Kinoshita et al. 2005 Pascreau et al. 2005). 

Kaempfer, R., 1984, Differential gene expression by messenger RNA competition for eukaryotic initiation factor 2, in Protein Synthesis Translation and Post-Translational Events (A. K. Abraham, T. S. Eikhom, and I. F. Pryme, eds.), pp. 57-76, The Humana Press, Clifton, New Jersey. 

Siekierka, J., Mitsui, K. I., and Ochoa, S., 1981, Mode of action of the heme controlled translational inhibitor Relationship of eukaryotic initiation factor 2-stim-ulating protein to translation restoring factor, Proc. Natl. Acad. Sci. USA 78 220. 

Many more protein factors are involved in eukaryotic initiation some systems contain more than 10 initiation factors. Particular features of translation initiation are also different in the higher organisms. Most notably, prokaryotic ribosomes can initiate internally on an mRNA (even on circular RNAs), while in eukaryotes a preinitiation complex binds to the 5 -end of the mRNA and then progresses to an initiation complex. Eukaryotic mRNAs are capped at their 5 -end with a 7-methylguanosine triphosphate structure, and one of the eukaryotic initiation factors binds this capped end. The preinitiation complex then moves along the mRNA and initiates translation at the first AUG codon it comes to. Consistent with this scanning mechanism is the observation that eukaryotic mRNAs do not contain Shine-Dalgarno-like sequences. 

In 1965, with a return to studies using natural mRNAs instead of synthetic polyribonucleotides, several investigators reported the requirement of a number of specialized protein factors for initiation of translation. They were found to be associated, in both prokaryotes and eukaryotes, with the small ribosomal subunit. Table 5 lists the eukaryotic initiation factors and their functions in this process. 

Genes of interest can be tagged at either the N or C terminal end. The decision to tag a protein at either the N or the C terminal depends upon the properties of the protein of interest. In our case, all the eukaryotic translation initiation factors were tagged C terminally to allow the endogenous promoter to influence the expression of the tagged protein. 

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