Transfer factor, ribosome specificity

As we have noted, the outcome of a virus infection is the synthesis of viral nucleic acid and viral protein coats. In effect, the virus takes over the biosynthetic machinery of the host and uses it for its own synthesis. A few enzymes needed for virus replication may be present in the virus particle and may be introduced into the cell during the infection process, but the host supplies everything else energy-generating system, ribosomes, amino-acid activating enzymes, transfer RNA (with a few exceptions), and all soluble factors. The virus genome codes for all new proteins. Such proteins would include the coat protein subunits (of which there are generally more than one kind) plus any new virus-specific enzymes. 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

The ribosome can carry two aminoacyl-tRNAs simultaneously. In the chain elongation stage, the growing polypeptide is carried on one of these tRNAs. The chain is transferred to the second tRNA, which adds its amino acid to the growing peptide, and displaces the first tRNA. The ribosome then moves one codon along the mRNA to allow the next to be read. Termination of protein synthesis involves the release of the completed polypeptide, expulsion of the last tRNA, and dissociation of the ribosome from the mRNA. This is signaled by specific termination codons (UAA, UAG, or UGA) in the mRNA and requires the participation of various release factors. 

Fig. 4. Role of the stop codon and lOSa-RNA in E. coli translation. (A) When a stop codon is encountered, a complex of two release factors, RF-1 and RF-3 or RF-2 and RF-3, binds instead of the tRNA. The release factor RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. The binding of the release factor complex results in hydrolysis of the peptidyl-tRNA and release of the peptide. (B) The role of lOSa-RNA. If truncated mRNA without a stop codon is translated in E. coli, the ribosome stops at the end of the mRNA. lOSa-RNA can then bind to the ribosomal A site and lOSa-RNA can act as tRNA by transferring an alanine to the truncated protein. Subsequently, lOSa-RNA acts as mRNA and a peptide tag with the indicated sequence is added to the truncated protein. lOSa-RNA encodes a stop codon and therefore the protein is released and then degraded by proteases specifically recognizing this C-terminal tag.

The chemical polymerization of even a moderately sized protein of a hundred amino acids in the laboratory is extremely laborious, and the yields of active product can often be low to zero (Kent and Parker, 1988). Cells accomplish this task by using an intricate mechanism which involves catalytic machinery composed of proteins, nucleic acids and their complexes, and synthesize polypeptide chains that are composed of hundreds of amino acids. This process is depicted in Fig. 2.4, and is described in the sections below. The basic components of the cellular protein synthesis apparatus, in all known biological systems, are ribosomes, which are aggregate structures containing over fifty distinct proteins, and three distinct molecules of nucleic acid known as ribosomal ribonucleic acid (ribosomal RNA or rRNA). The amino acids are brought to the ribosomes, the assembly bench , by an RNA molecule known appropriately as transfer RNA . Each of the twenty amino acids is specifically coupled to a set of transfer RNAs (discussed below) which catalyze their incorporation into appropriate locations in the linear sequence of polypeptide chains. Several other intracellular proteins known as init iation and elongation factors a re also required for protein synthesis. 

Translation ends when a termination codon enters the ribosomal A-site. Release of the newly synthesized protein chain from the ribosome is mediated by the action of the release factor RF-1 specific for the termination codons UAA and UAG or of the factor RF-2 specific for UAA and UGA. A third release factor and GTP stimulate this process. It is likely that the cleavage of tRNA from the completed protein chain is carried out by the same enzyme, namely the peptidyltransferase, which transfers the growing peptide chain from the P-site onto the aminoa< l-tRNA in the A-site. The termination process is completed by release of the newly synthesized protein chain, the de-ac lated tRNA and the messenger-RNA from the ribosome. Modification of the protein, e.g. acetylation and methylation, probably occurs when the growing chain is still on the ribosome and as soon as the amino acid to be modified become accessible for the appropriate non-ribosomal enzyme. 

Each time an additional peptide bond is formed, the growing polypeptide chain becomes linked to the last occurring transfer RNA fixed in the acceptor site of the ribosome. A translocation step which involves GTP hydrolysis to GDP and P then takes place. This step is catalysed by a specific enzyme (the translocase or G factor) from the supernatant During translocation, the peptidyl-tRNA reaches the second site C doQor ) on the ribosome thereby, the acceptor site for the interaction with the next aminoacyl-tRNA becomes free. This translocation implies the displacement of the ribosome relative to the messenger-RNA fibre. The peptide-bond formation reaction is then repeated (Fig. 8). 

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