Cytosolic tRNA

Retrieve one each of the cytosolic tRNA specific for the following amino acids from Saccharomyces cerevisiae ... 

In the rabbit the synthesis of cytosol tRNA is detectable at the 2-cell stage while formation of 28 S and 18 S RNA is found later (Morula to blastocyst transformation), quite opposite to the data reported for mouse preimplantation stages (Manes, 1971). Unfortunately, no data are available for mt RNA synthesis during these stages in rabbits. Therefore it may be wise not to extrapolate the exact sequence of events, even from one mammalian species to another. 

Mitochondria are unique organelles in that they contain their own DNA (mtDNA), which, in addition to ribosomal RN A (rRNA) and transfer RN A (tRNA)-coding sequences, also encodes 13 polypeptides which are components of complexes I, III, IV, and V (Anderson et al., 1981). This fact has important implications for both the genetics and the etiology of the respiratory chain disorders. Since mtDNA is maternally-inherited, a defect of a respiratory complex due to a mtDNA deletion would be expected to show a pattern of maternal transmission. However the situation is complicated by the fact that the majority of the polypeptide subunits of complexes I, III, IV, and V, and all subunits of complex II, are encoded by nuclear DNA. A defect in a nuclear-coded subunit of one of the respiratory complexes would be expected to show classic Mendelian inheritance. A further complication exists in that it is now established that some respiratory chain disorders result from defects of communication between nuclear and mitochondrial genomes (Zeviani et al., 1989). Since many mitochondrial proteins are synthesized in the cytosol and require a sophisticated system of posttranslational processing for transport and assembly, it is apparent that a diversity of genetic errors is to be expected. 

Not all the cellular DNA is in the nucleus some is found in the mitochondria. In addition, mitochondria contain RNA as well as several enzymes used for protein synthesis. Interestingly, mitochond-rial RNA and DNA bear a closer resemblance to the nucleic acid of bacterial cells than they do to animal cells. For example, the rather small DNA molecule of the mitochondrion is circular and does not form nucleosomes. Its information is contained in approximately 16,500 nucleotides that func-tion in the synthesis of two ribosomal and 22 transfer RNAs (tRNAs). In addition, mitochondrial DNA codes for the synthesis of 13 proteins, all components of the respiratory chain and the oxidative phosphorylation system. Still, mitochondrial DNA does not contain sufficient information for the synthesis of all mitochondrial proteins most are coded by nuclear genes. Most mitochondrial proteins are synthesized in the cytosol from nuclear-derived messenger RNAs (mRNAs) and then transported into the mito-chondria, where they contribute to both the structural and the functional elements of this organelle. Because mitochondria are inherited cytoplasmically, an individual does not necessarily receive mitochondrial nucleic acid equally from each parent. In fact, mito-chondria are inherited maternally. 

The formation of 0-seryl or 0-prolyl esters (Figure 1) of certain N-hydroxy arylamines has been inferred from the observations that highly reactive intermediates can be generated in vitro by incubation with ATP, serine or proline, and the corresponding aminoacyl tRNA synthetases (11,12,119). For example, activation of N-hydroxy-4-aminoquinoline-l-oxide (119,120), N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) to nucleic acid-bound products was demonstrated using seryl-tRNA synthetase from yeast or rat ascites hepatoma cells. More recently, hepatic cytosolic prolyl-, but not seryl-, tRNA synthetase was shown to activate N-hydroxy-Trp-P-2 (12) however, no activation was detectable for the N-hydroxy metabolites of AF, 3,2 -dimethyl-4-aminobiphenyl, or N -acetylbenzidine (122). 

The stem-loop structure in the noncoding 3 region of selenoprotein mRNAs has also been termed a SECTS element in mammals although it has a different overall structure. ° In silica analysis of the human genome sequence, using this consensus SECTS element along with the presence of the characteristic UGA codon within an exon, has led to the discovery of several new selenoproteins, including a selenium-dependent methionine sulfoxide reductase. It has been shown that a specific complex exists for selenoprotein synthesis that shuttles between the nucleus and the cytosol. This possibly protects the preformed complex for nonsense-mediated decay to allow for more efficient selenoprotein synthesis. The specific tRNA needed for selenocysteine... 

The existence of mitochondrial DNA, ribosomes, and tRNAs supports the hypothesis of the endosymbiotic origin of mitochondria (see Fig. 1-36), which holds that the first organisms capable of aerobic metabolism, including respiration-linked ATP production, were prokaryotes. Primitive eukaryotes that lived anaerobically (by fermentation) acquired the ability to carry out oxidative phosphorylation when they established a symbiotic relationship with bacteria living in their cytosol. After much evolution and the movement of many bacterial genes into the nucleus of the host eukaryote, the endosymbiotic bacteria eventually became mitochondria. 

The second key advance was made by Mahlon Hoagland and Zamecnik, when they found that amino acids were activated when incubated with ATP and the cytosolic fraction of liver cells. The amino acids became attached to a heat-stable soluble RNA of the type that had been discovered and characterized by Robert Holley and later called transfer RNA (tRNA), to form aminoacyl-tRNAs. The enzymes that catalyze this process are the aminoacyl-tRNA synthetases. 

Stage 2 Initiation The mRNA bearing the code for the polypeptide to be made binds to the smaller of two ri-bosomal subunits and to the initiating aminoacyl-tRNA. The large ribosomal subunit then binds to form an initiation complex. The initiating aminoacyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning of the polypeptide. This process, which requires GTP, is promoted by cytosolic proteins called initiation factors. 

During the first stage of protein synthesis, taking place in the cytosol, aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs. Each enzyme is specific for one amino acid and one or more corresponding tRNAs. Most organisms have one aminoacyl-tRNA synthetase for each amino acid. For amino acids with two or more corresponding tRNAs, the same enzyme usually aminoacylates all of them. 

The third stage of protein synthesis is elongation. Again, our initial focus is on bacterial cells. Elongation requires (1) the initiation complex described above, (2) aminoacyl-tRNAs, (3) a set of three soluble cytosolic proteins called elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), and (4) GTP. Cells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added. 

Elongation Step 3 Translocation In the final step of the elongation cycle, translocation, the ribosome moves one codon toward the 3 end of the mRNA (Fig. 27-25a). This movement shifts the anticodon of the dipeptidyl-tRNA, which is still attached to the second codon of the mRNA, from the A site to the P site, and shifts the de-acylated tRNA from the P site to the E site, from where the tRNA is released into the cytosol. The third codon of the mRNA now lies in the A site and the second codon in the P site. Movement of the ribosome along the mRNA requires EF-G (also known as translocase) and the energy provided by hydrolysis of another molecule of GTP. 

Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPj. The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites. In bacteria, the initiating aminoacyl-tRNA in all proteins is A-formylmethionyl-tRNAfMet. 

There are three major types of RNA that participate in the process of protein synthesis ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). They are unbranched polymers of nucleotides, but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a spe dfic amino acid to the site of protein synthesis. mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. The process of RNA synthesis is called transcription, and its substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase, which is a multisub-irit enzyme. In prokaryotic cells, the core enzyme has four subunits—... 

The sequences of all three pieces of RNA in the E. coli ribosomes are known as are those from many other species. These include eukaryotic mitochondrial, plas-tid, and cytosolic rRNA. From the sequences alone, it was clear that these long molecules could fold into a complex series of hairpin loops resembling those in tRNA. For example, the 16S rRNA of E. coli can fold as in Fig. 29-2A and eukaryotic 18S RNA in a similar way (Fig. 29-4).38/39/67 69 The actual secondary structures of 16S and 18S RNAs, within the folded molecules revealed by X-ray crystallography, are very similar to that shown in Fig. 29-2A. Ribosomal RNAs undergo many posttranscriptional alterations. Methylation of 2 -hydroxyls and of the nucleic acid bases as well as conversion to pseudouridines (pp. 1638-1641) predominate over 200 modifications, principally in functionally important locations that have been found in human rRNA.69a... 

Retrieve nucleotide sequences (fasta files) of yeast cytosolic and mitochondrial Gly-tRNA and submit them to RNA folding to obtain their secondary (cloverleal) structures and thermochemical data of foldings. 

Hashimoto T (1998) Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proc Natl Acad Sci USA 95 6860-6865 Hausmann A, Guilar Netz DJ, Balk J, Pierik AJ, Muhlenhoff U, Lill R (2005) The eukaryotic P loop NTPase Nbp35 an essential component of the cytosolic and nuclear iron-sulfur protein assembly machinery. Proc Natl Acad Sci USA 102 3266-3271 Horner DS, Foster PG, Embley TM (2000) Iron hydrogenases and the evolution of anaerobic eukaryotes. Mol Biol Evol 17 1695-1709... 

Cytosol Glycolysis, glycogenesis and glycogenolysis, hexose monophosphate pathway, fatty acid synthesis, purine and pyrimidine catabolism, aminoacyl-tRNA synthetases... 

Table 2IC50 (nM) values for selected a-aminoocylsulfamoyl-adenosines against plastidic and cytosolic oaTRS s (ND = Not Determined, LTRS = leucyl-tRNA synthetase, ITRS = isoleucyl-tRNA synthetase)... 

Dimethylallylpyrophosphate (DMAPP) originating from cytosolic mevalonate (JVIVA) pathway probably serves as a precursor of tRNA isoprenylation [80]. Release of cytokinins via tRNA degradation could at least serve as a possible source of cA-zeatin, which is much more abundant in RNA than the tran5-isomer [89] and further de novo biosynthesis of this cytokinin species [90] as well as its isomerization from trans-xQ-dim have not been proved yet. 

Thus, mitochondria require only 22 tRNA moleeules to read the genetic code as eompared to 31 required for the cytosolic system. 

Formylated methionyl-tRNA is important for initiation of translation Single mRNAs specify more than one gene product Cycloheximide blocks elongation during translation Cytosolic ribosomes are smaller than those found in prokaryotes Erythromycin inhibits elongation during translation... 

The answer is c. (Murray, pp 452—467. Scriver, pp 3—45. Sack, pp 1—40. Wilson, pp 101-120.) Prokaryotic ribosomes have a sedimentation coefficient of 70S and are composed of SOS and 30S subunits. Eukaryotic cytoplasmic ribosomes, either free or bound to the endoplasmic reticulum, are larger—60S and 40S subunits that associate to an SOS ribosome. Nuclear ribosomes are attached to the endoplasmic reticulum of the nuclear membrane. Ribosomes in chloroplasts and mitochondria of eukaryotic cells are more similar to prokaryotic ribosomes than to eukaryotic cytosolic ribosomes. Like bacterial ribosomes, chloroplast and mitochondrial ribosomes use a formylated tRNA. In addition, they are sensitive to many of the inhibitors of protein synthesis in bacteria. 

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