Ribosome eukaryotic proteins

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.

Genes can similarly be cloned and expressed in eukaryotic cells, with various species of yeast as the usual hosts. A eukaryotic host can sometimes promote post-translational modifications (changes in protein structure made after synthesis on the ribosomes) that might be required for the function of a cloned eukaryotic protein. 

An understanding of protein synthesis, the most complex biosynthetic process, has been one of the greatest challenges in biochemistry. Eukaryotic protein synthesis involves more than 70 different ribosomal proteins 20 or more enzymes to activate the amino acid precursors a dozen or more auxiliary enzymes and other protein factors for the initiation, elongation, and termination of polypeptides perhaps 100 additional enzymes for the final processing of different proteins and 40 or more kinds of transfer and ribosomal RNAs. Overall, almost 300 different macromolecules cooperate to synthesize polypeptides. Many of these macromolecules are organized into the complex three-dimensional structure of the ribosome. 

Ribosomal proteins Ribosomal proteins are present in considerably greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a number of roles in the structure and function of the ribosome and its interactions with other components of the translation system. 

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... 

The first success was demonstration in 1994, with the report of a large, diverse library of decapeptides displayed and selected while associated with E. coli S30 polysomes and RNA.262 The key to Dower s success was the application of natural product antibiotics that were known to interfere with protein synthesis by stabilizing the ribosome-mRNA-protein complex. Thus, rifampicin and chloramphenicol (for prokaryotic system) or cycloheximide (for eukaryotic system) were used.2 3 Because these antibiotics halt the translation at random locations, the ensuing libraries were composed of mostly truncated peptides and thus not really suitable for the generation of cDNA libraries. Later, removal of the stop codon from mRNA was used to stall the translation at the end of the mRNA.264,265 Several improvements have been made more recently to stabilize the... 

Shiga toxin produced by Shigella dysenteriae has similar structural features. The toxin binds to a glycolipid (Gb3), undergoes endocytosis, and the enzymatie Ai fragment, which is a specific N-glycosidase, removes adenine from one particular adenosine residue in the 28S RNA of the 60S ribosomal subunit. Removal of the adenine inactivates the 60S ribosome, blocking protein synthesis. Ricin, abrin, and a number of related plant proteins inhibit eukaryotic protein synthesis in a similar manner (Chapter 25). 

RFl and RF2 are compact proteins that in eukaryotes resemble a tRNA molecule. When bound to the ribosome, the proteins unfold to bridge the gap between the stop codon on the mRNA and the peptidyl transferase center on the SOS subunit. Although the precise mechanism of release is not known, the release factor may promote, assisted by the peptidyl transferase, a water molecule s attack on the ester linkage, freeing the polypeptide chain. The detached polypeptide leaves the ribosome. Transfer RNA and messenger RNA remain briefly attached to the 70S ribosome until the entire complex is dissociated in a GT F-dependent fashion in response to the binding of EF-G and another factor, called the ribosome release factor (RRF) (Figure 30.25). 

In eukaryotic cells, a ribosome remains free in the cytoplasm unless it is directed to the endoplasmic reticulum (ER), the extensive membrane system that comprises about half the total membrane of a cell. The region that binds ribosomes is called the rough ER because of its studded appearance, in contra.sl with the smooth ER, which is devoid of ribosomes (Figure 30.28). Free ribosomes synthesize proteins that remain within the cell, either within the cytoplasm or directed to organelles bounded by a double membrane, such as the nucleus, mitochondria and chloroplasts. Ribosomes bound to the ER usually synthesize proteins destined to leave the cell or to at least contact the cell exterior from a position in the cell membrane. These proteins fall into three major classes secretory proteins (proteins exported by the cell), lysosomal proteins, and proteins spanning the plasma membrane. Virtually all integral membrane proteins of the cell, except those located in the membranes of mitochondria and chloroplasts, are formed by ribosomes bound to the ER. 

Endo, Y., Tsurugi, K. and Lamberf, J.M. (1988a) The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes the RNA A-glycosidase activity of the proteins. Biochem Biophys Res Commun, 150, 1032-1036. 

Ribosomal RNA (rRNA) is a component of the ribosomes, the protein synthesis factories in the cell. rRNA molecules are extremely abundant, making up at least 80 percent of the RNA molecules found in a typical eukaryotic cell. Virtually all ribosomal proteins are in contact with rRNA. Most of the contacts between ribosomal subunits are made between the 16S and 23 S rRNAs such that the interactions involving rRNA are a key part of ribosome function. The environment of the tRNA-binding sites is largely determined by rRNA. The rRNA molecules have several roles in protein synthesis. 16S rRNA plays an active role in the functions of the 308 subunit. It interacts directly with mRNA, with the 508 subunit, and with the anticodons of tRNAs in the P- and A-sites. Peptidyl transferase activity resides exclusively in the 238 rRNA. Finally, the rRNA molecules have a structural role. They fold into three-dimensional shapes that form the scaffold on which the ribosomal proteins assemble. 

NygSrd, O. Nilsson, L. (1990). Translational dynamics. Interactions between the translational factors, tRNA and ribosomes during eukaryotic protein synthesis. Eur. J. Biochem. 191,1-17. 

The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5 capped end as just described. However, some cellular mRNAs contain an internal ribosome entry site (IRES) located far downstream of the 5 end. In addition, translation of some viral mRNAs, which lack a 5 cap, is initiated at IRESs by the host-cell machinery of infected eukaryotic cells. Some of the same translation initiation factors that assist in ribosome scanning from a 5 cap are required for locating an internal AUG start codon, but exactly how an IRES is recognized is less clear. Recent results indicate that some IRESs fold into an RNA structure that binds to a third site on the ribosome, the E site, thereby positioning a nearby internal AUG start codon in the P site. 

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