Ribosome initiation sites

Very recently, a third hypothesis has been pubHshed. Morita and co-workers [47] have suggested that the rpoH mRNA secondary structure itself acts as a thermosensor. In the absence of heat stress, the rpoH mRNA is folded into a secondary structure that occludes the ribosome binding site and the initiation codon. Upon heat shock, this structure is unfolded allowing ribosome binding and enhanced synthesis. 

In contrast to most mRNAs, which become untranslatable after a temperature downshock, cold shock mRNAs possess a mechanism to form the translation initiation complex at low temperature without cold shock ribosomes. A close inspection of the mRNAs of class I cold shock proteins reveal that they are equipped with an extra ribosome-binding site called the downstream box located within the coding region of their transcript [130]. It would be interesting to know whether introduction of this downstream box into a cellular mRNA would convert it into a transcript which can be transcribed immediately after a cold shock. In the case of the cspA mRNA it has been shown that in the absence of the downstream box the initiation complex cannot be formed at low temperature during the accHmation phase [131]. 

The process of RNA synthesis in bacteria—depicted in Figure 37-3—involves first the binding of the RNA holopolymerase molecule to the template at the promoter site to form a PIC. Binding is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine) then associates with the initiation site on the 3 subunit of the enzyme. In the presence of the appropriate nucleotide, the RNAP catalyzes the formation of a phosphodiester bond, and the nascent chain is now attached to the polymerization site on the P subunit of RNAP. (The analogy to the A and P sites on the ribosome should be noted see Figure... 

Figure 38-10. Picornavimses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of viral mRNAs. To gain selective advantage, certain viruses (eg, poliovirus) have a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap. The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example.

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... 

Fig. 24.6 The use of a vector carrying a promoter and adjacent ribosome binding site (RBS) and initiation codon to obtain synthesis of proinsulin from a synthetic gene. The arrow indicates the direction of transcription.

Table 6.1 Translation initiation factor requirements of internal ribosome entry site (IRES) elements... 

In the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

Lomakin, I. B., Hellen, C. U., and Pestova, T. V. (2000). Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol. Cell Biol. 20, 6019-6029. 

Pisarev, A. V., Shirokikh, N. E., and Hellen, C. U. (2005). Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C R Biol. 328, 589-605. 

Kolupaeva, V.G., Fomakin, I.B., Pestova, T.V., and Hellen, C.U.T. (2003) Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocar-ditis virus internal ribosomal entry site. Mol. Cell. Biol. 23, 687-698. 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

Generation of the initiator N-formyl-> movement of the uncharged tRNA into the ribosomal E site (before... 

Many viral RNAs that are formed within eukaryotic cells lack a 5 cap. They depend upon internal ribosomal entry sites (IRESs). This has been studied most with picoma viruses.338 502 503 These viruses not only initiate translation at discrete sites in uncapped RNA but carry out a proteolytic cleavage of initiation factor 4G (Fig. 29-11), which seems to be necessary for initiation of viral-RNA translation.338 504 The IRES... 

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