Eukaryotes ribosomes

SERPR, serine protease SialylT, sialyltransferase SLOX, soya bean 15-lipoxygenase, soya bean lipoxygenase SNF1K, SNF1 protein kinase kinase 80S PT, 80S ribosome (eukaryote) peptidyl transferase... 

Ribosomes. Eukaryotic ribosomes are larger. They consist of a 60S large subunit and a 40S small subunit, which come together to form an SOS particle having a mass of 4200 kd, compared with 2700 kd for the prokaryotic 70S ribosome. The 40S subunit contains an 18S RNA that is homologous to the prokaryotic 16S RNA. The 60S subunit contains three RNAs the 5S and 28S RNAs are the counterparts of the prokaryotic 5S and 23S molecules its 5.8S RNA is unique to... 

In contrast, RNA occurs in multiple copies and various forms (Table 11.2). Cells contain up to eight times as much RNA as DNA. RNA has a number of important biological functions, and on this basis, RNA molecules are categorized into several major types messenger RNA, ribosomal RNA, and transfer RNA. Eukaryotic cells contain an additional type, small nuclear RNA (snRNA). With these basic definitions in mind, let s now briefly consider the chemical and structural nature of DNA and the various RNAs. Chapter 12 elaborates on methods to determine the primary structure of nucleic acids by sequencing methods and discusses the secondary and tertiary structures of DNA and RNA. Part rV, Information Transfer, includes a detailed treatment of the dynamic role of nucleic acids in the molecular biology of the cell. 

FIGURE 11.25 The organization and composition of prokaryotic and eukaryotic ribosomes. 

Ribosomes are ancient ribonucleoprotein complexes that are the sites of protein synthesis in living cells. Their core structures and fundamental functional mechanisms have been conserved throughout the three domains of life bacteria, archaea and eukaryotes. All ribosomes are organized into two subunits that are defined by their apparent sedimentation coefficient, measured in Svedberg units (S). There is a general... 

Not only eukaryotic cells but also bacteria have successfully been targeted by PNA anhsense strategies. Thus it has been shown that PNA complementary to ribosomal RNA or mRNA encoding an essential fatty acid biosynthesis protein, effectively kills E. coli. Furthermore, it has been shown that PNA directed to the start codon of the y -lactamase gene re-sensitized otherwise resistant E. coli to the antibiohc ampiciUin [64—66]. Conjugating a simple transporter peptide to the PNA increased the potency significantly, and an even more potent antibacterial PNA... 

In all prokaryotic and eukaryotic organisms, three main classes of RNA molecules exist messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA... 

In addition to the catalytic action served by the snRNAs in the formation of mRNA, several other enzymatic functions have been attributed to RNA. Ribozymes are RNA molecules with catalytic activity. These generally involve transesterification reactions, and most are concerned with RNA metabofism (spfic-ing and endoribonuclease). Recently, a ribosomal RNA component was noted to hydrolyze an aminoacyl ester and thus to play a central role in peptide bond function (peptidyl transferases see Chapter 38). These observations, made in organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme. This has revolutionized thinking about enzyme action and the origin of life itself. 

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 charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

Ribosomes in bacteria and in the mitochondria of higher eukaryotic cells differ from the mammalian ribosome described in Ghapter 35. The bacterial ribosome is smaller (70S rather than SOS) and has a different, somewhat simpler complement of RNA and protein... 

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

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 7.4 (a) IREs in eukaryotic mRNAs the secondary structures of ferritin and transferrin receptor IREs. (b) The IRE localization in mRNAs the translation/ribosome binding element in the 5 -UTR of ferritin mRNA is above, that of the stability/ turnover element in the 3 -UTR of transferrin receptor mRNA is below. Adapted from Theil, 1998, by courtesy of Marcel Dekker, Inc. 

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.

Ribosomal RNA Different from eukaryotes Different from eukaryotes... 

Singh, C. R., He, H., Ii, M., Yamamoto, Y., and Asano, K. (2004). Efficient incorporation of eukaryotic initiation factor 1 into the multifactor complex is critical for formation of functional ribosomal preinitiation complexes in vivo.J. Biol. Chem. 279, 31910-31920. 

The field of translation initiation has focused on the initial round ofribosomal subunit recruitment to an mRNA. Presumably, these events are mirrored in the subsequent rounds of initiation necessary for polyribosome formation. Importantly, because mRNAs are typically present in large polyribosomes (averaging 9-13 ribosomes per mRNA), the initiation events that govern ribosome recruitment to preexisting polyribosomes constitute the majority of translation initiation cycles occurring in an mRNA s lifetime. Whether or not these initiation events mimic the first round of initiation is not yet known. Since eukaryotic cells divide ribosomes between two subcellular compartments, the cytosol and ER membrane, it is also important to know if the mechanism of translation initiation on ER-bound ribosomes is similar to that occurring on soluble ribosomes and, importantly, whether ER-bound ribosomes can direcdy (re) initiate translation on bound polyribosmes. 

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