Factor puromycin

One type of fatty liver that has been smdied extensively in rats is due to a deficiency of choline, which has therefore been called a lipotropic factor. The antibiotic puromycin, ethionine (a-amino-y-mercaptobu-tyric acid), carbon tetrachloride, chloroform, phosphorus, lead, and arsenic all cause fatty liver and a marked reduction in concentration of VLDL in rats. Choline will not protect the organism against these agents but appears to aid in recovery. The action of carbon tetrachloride probably involves formation of free radicals... 

Demeuse P, Fragner P, Leroy-Noury C et al. (2004) Puromycin selectively increases Mdrla expression in immortalized rat brain endothelial cell lines. J Neurochem 88 23-31 Gospodarowicz D, Massoglia S, Cheng J, Fujii DK (1986) Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries. J Cell Physiol 127 121-136... 

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

Mach, Reich and Tatum were able to demonstrate an inhibition of the biosynthesis of protein in cells of B. brevis by chloramphenicol and puromycin without affecting the synthesis of tyrocidine . Several analogues of amino acids were found, which inhibited the biosynthesis of tyrocidine without affecting that of protein and vice versa. In contrast to protein synthesis, the production of tyrocidine did not depend on the continuous synthesis of RNA. Furthermore, environmental factors were able to control the relative amounts of the different tyrocidines synthesized by genetically homogeneous cultures . Addition of phenylalanine to the culture medium resulted in the almost exclusive synthesis of tyrocidine A, whereas the unsupplemented culture produced tyrocidine A, B and C. In the presence of tryptophan, a new form of tyrocidine, called tyrocidine D, containing three tryptophan in place of three phenylalanine residues, was produced. This lack of absolute requirement for specific amino acids in the formation of a peptide bond is in contrast to the strict specificity of sequential incorporation of amino acids... 

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 mechanism of action of blasticidin S has been studied for more than 20 years. In the 1960s, it was recognized that blasticidin S stimulates T-factor-dependent binding of phenylalanine tRNA to ribosomes (96), and that it inhibited the effects of puromycin in a manner similar to chloramphenicol (97). Blasticidin S binds strongly to the SOS sub-... 

Whenever tested, binding of labels to the ribosome has been shown to be stimulated severalfold by the appropriate or by initiation factors. -" Based on reactivity with puromycin, all pep-tidyl-tRNA probes tested were considered to occupy predominantly the ribosomal donor site. This conclusion was supported by the observed ability of several Phe-tRNA derived probes to form a peptide bond with added Phe-tRNA as acceptor. - - Similarly, modified Met-tRNA bound with f2-RNA has been shown to interact with Ala-tRNA to form the Met-Ala initiation sequence typical of the phage coat protein. ... 

Photoaffinity Labeling of Ribosomes with the Unmodified Ligands Puromycin and Initiation Factor 3... 

Initiation of the translation is well known in E. coli. The first step, promoted by a proteic factor (F3 or B) is the formation of a complex between a 30 S ribosomal subparticle and the initiation site of a messenger RNA (AUG codon for methionine). One particular species of Met-tRNA , on which the NH2 group of methionine may be formylated after transfer-RNA acylation, associates to this complex if other factors (F2 and Fi, or C and A) and GTP are present (GTP is included in the resulting complex). An entity called complex I is thus formed, and is then completed to complex II by addition of a SOS ribosomal particle. In this complex II, formylmet-tRNA Ms bound to the A (acceptor) site on the ribosome. The last step in the initiation process, which is catalysed by the F2 factor and which involves GTP hydrolysis to GDP and Pi, is the translocation of the formyl-met-tRNA to the P (donor) site on the ribosome in this way the so-called complex III is formed. (Ilie A and P sites on the ribosome were defined by using the property of puromycin to only react with a peptidyl-tRNA if this entity is at the donor (P) site.) The precise role of the different initiation factors which are obtained from the ribosome wash is not yet completely established. 

GTP hydrolysis must have occurred. Indeed, nonhydrolyzable analogues of GTP block joining of the 60 S subunit (Trachsel et al., 1977 Benne and Hershey, 1978). These reactions require the presence of eIF-5, the 60 S joining factor (160,000 Mr) (Trachsel et al., 1977 Benne and Hershey, 1978). The resulting 80 S initiation complex is apparently free of initiation factors, and its Met-tRNAf is reactive to puromycin, meaning that it is capable of entering the elongation phase of polypeptide synthesis. 

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