Ribosomes into Subunits

Another initiation factor, eIF-4C (17,000 Mr) also binds to 40 S subunits and somewhat reduces their association with 60 S subunits (Thomas et al., 1980a). 

Binding of mRNA to the 40 S ribosomal subunit cannot take place unless the initiator tRNA, Met-tRNAf, is first bound (Schreier and Staehelin, 1973 Darnbrough et al., 1973). This means that the recognition and binding of Met-tRNAf are an integral part of the mRNA-binding process. 

As will be reviewed in detail in Section 5.1, eIF-2, in addition to binding Met-tRNAf, can bind to mRNA (Kaempfer, 1974 Kaempfer et al., l97Sa,b Barrieux and Rosenfeld, 1978). The GTP-dependent binding of Met-tRNAf to eIF-2 is inhibited competitively by mRNA (Kaempfer et al., 1978 Barrieux and Rosenfeld, 1978 Rosen et al., 1981a Chaudhuri et al., 1981). In the presence of eIF-2A, however, this inhibition of ternary complex formation by mRNA is less pronounced (Roy et al., 1981). Possibly, therefore, eIF-2A may act to prevent the interaction between mRNA and eIF-2 until after ternary complex formation has occurred. 

More recently, it was found that phosphorylation of the a-subunit of eIF-2 prevents the GDP/GTP exchange reaction and hence the recycling of eIF-2 in translation (Clemens et al., 1982 Siekierka et al., 1982 see Section 7). That suggests that the a-subunit of eIF-2 may interact with eIF-2B, and this, in turn, fits the finding that the a-subunit binds guanine nucleotides (Barrieux and Rosenfeld, 1977). 

Met-tRNAf, on the other hand, is bound by the 3-subunit of eIF-2 (Barrieux and Rosenfeld, 1977 Nygard et al., 1980). 

---

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. 

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. 

After dissociation of the 70 S ribosome into its two subunits followed by zonal centrifugation for the separation and isolation of the 30 S and 50 S subunits on a preparative scale, the ribosomal proteins were extracted by acetic acid and then separated by cellulose ion exchange chromatography and by gel filtration on Sephadex in the presence of 6 M urea. In this way all the 53 individual ribosomal proteins have been isolated (Wittmann, 1974). Proteins prepared in this manner have been used for physical studies (Brimacombe et al., 1978 Wittmann, 1982) as well as for immunological investigations (Stoffler et al., 1980 Lake,... 

S ribosomes into their 40S and 60S subunits. This depends upon the 700-kDa eIF3, a complex of 5-11 peptides of mass 30 to 170 kDa each, which binds to the 40S subunit (Fig. 29-11, step a). 306,311,318-320 jn a sepa ... 

The ribosome structure is perhaps the most impressive natural rotaxane. Messenger RNA, as the rotaxane thread, is clamped by the ribosomal protein subunits which read each codon and transcribe it into a sequence of amino acids that are introduced by transfer RNAs to build up the desired protein. The importance of the ribosomes has been reflected in the number of Nobel Prize recipients associated with their discovery (Palade in 1974) and elucidation of their structures and functions (Ramakrishnan, Steitz and Yonath in 2009). 

Halobacterial ribosomes are a special case they exist as 70 monomers in the presence of 100mM Mg " and near to saturating concentrations (3.1-4.0M) of K ions [67,68] and dissociate progressively upon lowering the Mg concentration [68] complete dissociation of 708 monomers into subunits that are synthetically active [69] occurs only upon exposure to a tenfold lower concentration of Mg " ions in the presence of a stabilizing (3.1 M) concentration of monovalent (K ) cations [67,68]. 

Peptidyltransferase assays have also provided insight into the mechanisms whereby spermine promotes, and NHJ ions inhibit, polypeptide synthesis on the ribosomes of S. solfataricus, T. tenax and D. mobilis (see section 3.3). First, the 30S uncoupled peptidyltransferase activity is absolutely dependent on spermine while being totally unaffected by monovalent cations. Secondly, monovalent cations strongly inhibit the 30S subunit coupled reaction [66]. Thus, polyamines appear to be obligatorily required to convert the catalytic center of the spermine-dependent ribosomes into an active conformation, whereas monovalent cations inhibit polypeptide synthesis by preventing 30S subunits from interacting with the cognate SOS particles (ref. [66], see below). 

Altamura et al. [178] and Londei et al. [179] investigated the ability of SOS and 30S subunits from phylogenetically disparate archaea to form synthetically active hybrid ribosomes with subunits from bacteria and eucarya, in the presence of Mg " concentrations (lS-18mM) which are optimal for polyphenylalanine synthesis. With poly(U) as the template and Phe-tRNA (or [jV-acetyl-Phe]-puromycin) as the substrate, SOS and 30S subunits from Euryarchaeota (M vannielii) and Crenarchaeota (5. solfataricus) could be assembled into hybrid active monosomes in all reciprocal combinations surprisingly, however, both reciprocal combinations of archaeal (S. solfataricus, M. vannielii) and eucaryal S. cerevisiae) ribosomal subunits gave rise... 

The dissociation of the inactive ribosome into its subunits is dependent on the activity of two initiation factors, eIF-3 and eIF-6, which keep subunits apart by binding to the 40S and the 60S subunits, respectively (Hershey, 1991 Rhoads, 1991 Merrick, 1992). eIF-3 is the largest of the initiation factors (molecular mass 600-650 kDa) comprising 8-10 different polypeptides. In comparison, eIF-6 is a single polypeptide of about 25 kDa. 

The ribosome provides an easily accessible source of endogenous RNA-protein complexes that participate in the overall process of translation. In this section, we present protocols for obtaining salt-washed ribosomes from E. coli and mammalian cell-lines. However, if highly active vacant couples devoid of tRNAs and mRNAs is the aim, it is necessary to use more elaborate protocols that include dissociation of bacterial 70 S tight couples into subunits,17... 

TERMINATION In eukaryotic cells two releasing factors, eRF-1 and eRF-3 (a GTP-binding protein), mediate the termination process. When GTP binds to eRF-3, its GTPase activity is activated. eRF-1 and eRF-3-GTP form a complex that bind in the A site when UAG, UGA, or UAA enter. Then GTP hydrolysis promotes the dissociation of the releasing factors from the ribosome. This step is soon followed by the release of mRNA and the separation of the functional ribosome into its subunits. As described, the release of the newly synthesized polypeptide is catalyzed by peptidyl transferase. 

Two initiation factors (IFl and IF3) bind to a VOS ribosome. IF3 and IFl appear to promote the dissociation of VOS ribosomes into free 30S and SOS subunits. mRNA and a third initiation factor (IF2), which carries a molecule of GTP and the charged initiator tRNA bind to a free 30S subunit. IF2 is one of a class of G proteins (see here). After these have all bound, the 30S initiation complex is complete. 

Prokaryotic initiation factors. In addition to the ribosomal proteins, the initiation factors IFl, IF2, and IF3, whose molecular masses are 9.5, 9.7, and 19.7 kDa, respectively, " are essential. They coordinate a sequence of reactions that begins with the dissociation of 70S ribosomes into their 30S and 50S subunits. Then, as is shown in Fig. 29-10, the mRNA, the initiator tRNA charged with formylmethionine, the three initiation factors, and the ribosomal subunits react to form 70S programmed ribosomes, which carry the bound mRNA and are ready to initiate protein synthesis. IF2 is a specialized G protein (Chapter 11), which binds and hydrolyzes GTP. It resembles the better known elongation factor EF-Tu (Section 2). The 172-residue IF3 consists of two compact a/p domains linked by a flexible sequence, which may exist as an a Its C-terminal domain binds to the central domain of the 16S RNA near nucleotides 819-859 (Fig. 

FIGURE 46-1 Inhibition of bacterial protein synthesis by tetracyclines. Messenger RNA (mRNA) attaches to the 30S subunit of bacterial ribosomal RNA. The P (peptidyl) site of the 50S ribosomal RNA subunit contains the nascent polypeptide chain normally, the aminoacyl tRNA charged with the next amino acid (aa) to be added to the chain moves into the A (acceptor) site, with complementary base pairing between the anticodon sequence of tRNA and the codon sequence of mRNA. Tetracyclines inhibit bacterial protein synthesis by binding to the 30S subunit and blocking tRNA binding to the A site. 

FIGURE 12.17 Simultaneous protein synthesis on polysomes. A single mRNA molecule is translated by several ribosomes simultaneously. Each ribosome produces one copy of the polypeptide chain specified by the mRNA. When the protein has been completed, the ribosome dissociates into subunits that are used in further rounds of protein synthesis. 

Initiation must start wiith a small ribosomal subunit 808 or 70S ribosomes are inactive and their spontaneous dissociation into subunits under physio-... 

Initiation factor 3 (IF-3) is one of three protein cofactors necessary for polypeptide chain initiation. It is now known that such initiation proceeds via dissociation of 70 S ribosomes into 30 S and 50 S subunits. IF-3, by binding tightly to the 30 S subunit, strongly favors this dissociation, and also appears to have the additional function of directing the binding of 30 S ribosomal subunits to start signals in messenger RNA. Recently, a structurally detailed hypothetical mechanism account-... 

Ribosomes are more or less round organelles found in the cytoplasm, plastids and mitochondria, which consist of roughly 60% RNA and 40% protein. The well-studied ribosomoes of pea seedlings possess the shape of a spheroid with axes 250 and 160 A long. Their sedimentation constant is 80 S, somewhat larger than that of the E. coli ribosomes (70 S) which have also been well-studied. If a suspension of ribosomes is deprived of each dissociates into subunits, one of 60 S and one of 40 S (in the case of E. coli, 50 S and 30 S). The RNA of the ribosomes is designated rRNA (ribosomal RNA). It can constitute over 90% of the total cellular RNA. However, very little is known with certainty about its function. 

Antibiotic A201A. Antibiotic A201A (23), produced by S. capreolus is an /V -dimethyladenine nucleoside stmcturaHy similar to puromycin (19). Compound (23) which contains an aromatic acid and monosaccharide residues (1,4), inhibits the incorporation of amino acids into proteins but has no effect on RNA or DNA synthesis. Compound (23) does not accept polypeptides as does (19), and does appear to block formation of the initiation complex of the SOS subunit. It may block formation of a puromycin-reactive ribosome. 

---