Translocation translation reactions

The final step in elongation is known as translocation (fig. 29.17). This reaction, like aminoacyl-tRNA binding, is catalyzed by a factor (the translocation factor, known as EF-G in prokaryotic systems and EF-2 in eukaryotic systems) that cycles on and off the ribosome and hydrolyzes GTP in the process. The overall purpose of translocation is to move the ribosome physically along the mRNA to expose the next codon for translation. 

DNA polymerase and 5 - 3 exonuclease functions of the enzyme. Nucleotide hydrolysis in the 5 - 3 direction concomitant with nucleotide polymerization results in translocation of the position of the discontinuity by a process termed nick translation (Fig. 2) (30). Discontinuities or nicks in DNA can be introduced into intact DNA by limited digestion with pancreatic DNase I, which generates 3 -hydroxyl termini in double-stranded DNA. If radioactive nucleotides are used in the reaction with DNA polymerase 1, randomly and uniformly labeled DNA is produced (31). 

No matter what the organism, translation consists of three phases initiation, elongation, and termination. The elongation reactions, which include peptide bond formation and translocation, are repeated many times until a stop codon is reached. Posttranslational reactions and targeting processes vary according to cell type. 

The overall reaction carried out by BiP is an Important example of how the chemical energy released by the hydrolysis of ATP can power the mechanical movement of a protein across a membrane. Some bacterial cells also use an ATP-driven process for translocating completed proteins across the plasma membrane. However, the mechanism of post-translational translocation in bacteria differs somewhat from that in yeast, as we describe in Section 16.4. 

Inhibitors of translation - A number of the common inhibitors of prokaryotic translation are also effective in eukaryotic cells. They include pactamycin, tetracycline, and puromycin. Inhibitors that are effective only in eukaryotes include cycloheximide and diphtheria toxin. Cycloheximide inhibits the peptidyltransferase activity of the eukaryotic ribosome. Diphtheria toxin is an enzyme, coded for by a bacteriophage that is lysogenic in the bacterium Corynebacterium diphtheriae. It catalyzes a reaction in which NAD+ adds an ADP ribose group to a specially modified histidine in the translocation factor eEF2, the eukaryotic equivalent of EF-G (Figure 28.36). Because the toxin is a catalyst, minute amounts can irreversibly block a cell s protein synthetic machinery. As a result, pure diphtheria toxin is one of the most deadly substances known. 

The statement that not the compound itself but its biosynthetic route has the taxonomical sig ni cance may be still valid. However, we know already that the biosynthetic route consists of complex metabolomic process including the genomic constitution, gene expression, transcriptional and translational regulation, enzyme activities at different biosynthetic levels, interactions with transporters, translocation, and spatial isolation (Dudareva et al., 2006 Dinesh and Nagegowda, 2010). The result of the enzymatic reaction is determined not only by the availability and constitution of the enzyme but also by the availability of precursors and different interactions therefore, the simple presence or absence of a certain enzyme in general cannot determine the nal product. 

Nick translation is one of the DNA-labeling techniques conventionally used to prepare hybridization probes. Nick translation utilizes combined activities of DNase I which introduces nicks (or single-strand breaks) and the 5 -(exo)nuclease and polymerase activities of E. coli DNA Pol I. While the 5 -nuclease activity of Pol I removes the nucleotides from the 5 -phosphoryl terminus, the polymerase activity carries out the sequential addition of nucleotides to the 3 -hydroxyl terminus, thus translocating the nick. When a highly radioactive nucleotide, e.g., [o - P]dATP, is included during the reaction, nick translation results in the uniform labeling of duplex molecules with a specific activity >10 cpm//zg DNA (1). Nick translation produces labeled DNA probes from both strands. Pol Ik, which lacks 5 -nuclease activity, cannot perform the nick translation, but it can carry out a strand displacement synthesis. 

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