Isopentenyl tRNA

Seif, E., and Hallberg, B. M. (2009). RNA-protein mutually induced fit structure of Escherichia coli isopentenyl-tRNA transferase in complex with tRNA(Phe). J. Biol. Chem. 284, 6600—6604. 

Cholesterol biosynthesis proceeds via the isoprenoids in a multistep pathway. The end product, cholesterol, and the intermediates of the pathway participate in diverse cellular functions. The isoprenoid units give rise to dolichol, CoQ, heme A, isopentenyl-tRNA, famesylated proteins, and vitamin D (in the presence of sunlight and 7-dehydrocholesterol). Dolichol is used in the synthesis of glycoproteins, CoQ in the mitochondrial electron transport chain, famesylation and geranylgeranylation by posttrans-lational lipid modification that is required for membrane association and function of proteins such as p2V and G-protein subunits. 

In some cases, an isoprene residue is used as an element to modify molecules chemically. One example of this is N -isopentenyl-AMP, which occurs as a modified component in tRNA. 

Cytokinins are unique among plant hormones in that adenine compounds of identical structure occur in nucleic acids. More specifically, particular cytokinin-active ribonucleosides occur as components of certain molecular species of tRNA. Zachau et al. (19), during the determination of the base sequences of serine tRNA in yeast, first reported an "odd" base immediately adjacent to the 3 end of the anticodon. In collaboration with Biemann et al. (20), this "odd" base was identified as the natural cytokinin isopentenyl-adenosine, which is one of the most highly active cytokinins known. 

Figure 5 Structural features of the tRNA from E. coli. Elements involved in recognition by seryl-tRNA synthetase are shaded, antideterminants against recognition by EF-Tu are hatched. The modified bases are D dihydrouridine F pseudouridine i A isopentenyl-adenosine T ribothymidine. Tertiary interactions involving base pairing are indicated by lines, those with intercalations by arrows...

The enzyme A -isopentenyl pyrophosphate tRNA-A -isopentenyl-transferase from Escherichia coli was shown to have a molecular weight of about 55 000. 

In contrast to s U and mnm s U biosyntheses, it is thought that ms i A and s C biosyntheses follow a [4Fe-4S] cluster-dependent pathway. The modification of ms i A has been found at position 37 of tRNAs and is believed to be a multistep reaction, where A -isopentenylation (discussed in the alkylation section) takes place first followed by thiolation and methylation. It has been shown that thiolation and methylation in ms i A biosynthesis are catalyzed by the proteins IscS and MiaB. Simultaneously, it was reported by two distinct groups that IscS is involved in the biosyntheses of all identified thiolated nucleosides however, unlike the cases of s U and mnm s U mentioned earlier in this section, IscS delivers the sulfur to IscU, a scaffold protein with a [4Fe S] cluster, which in turn facilitates the cluster assembly. Even though the mechanism has yet to be elucidated in detail, the [4Fe-4S] cluster can then be transported to activate the apo-form of a [4Fe-4S] protein. 

Dimethylallylation of adenosine (i A) refers to the modification occurring at position A37 (3 adjacent to the anticodon) in both prokaryotic and eukaryotic tRNAs. In some of the early literature, this reaction was also known as isopentenylation, while i A was termed A -isopentenyladenosine. Several decades ago, i A was first isolated from yeast. In many organisms, it can be further thiomethylated at to form ms i A, whose biosynthetic mechanism had been discussed earlier in this chapter. i A biosynthesis is catalyzed by dimethylallyltransferase, which is encoded by the miaA gene in E. and the modS gene in yeast. °... 

Essential non-steroidal isoprenoids, such as dolichol, prenylated proteins, heme A, and isopentenyl adenosine-containing tRNAs, are also synthesized by this pathway. In extrahepatic tissues, most cellular cholesterol is derived from de novo synthesis [3], whereas hepatocytes obtain most of their cholesterol via the receptor-mediated uptake of plasma lipoproteins, such as low-density lipoprotein (LDL). LDL is bound and internalized by the LDL receptor and delivered to lysosomes via the endocytic pathway, where hydrolysis of the core cholesteryl esters (CE) occurs (Chapter 20). The cholesterol that is released is transported throughout the cell. Normal mammalian cells tightly regulate cholesterol synthesis and LDL uptake to maintain cellular cholesterol levels within narrow limits and supply sufficient isoprenoids to satisfy metabolic requirements of the cell. Regulation of cholesterol biosynthetic enzymes takes place at the level of gene transcription, mRNA stability, translation, enzyme phosphorylation, and enzyme degradation. Cellular cholesterol levels are also modulated by a cycle of cholesterol esterification mediated by acyl-CoA cholesterol acyltransferase (ACAT) and hydrolysis of the CE, by cholesterol metabolism to bile acids and oxysterols, and by cholesterol efflux. 

Tricyclic hypermodified nucleosides are found in archaeal and eukaryotic tRNAs and are frequently observed at position 34 (wobble base) or position 37 (adjacent to the anticodon). Position 37 typically contains a hypermodified nucleoside such as N -threonylcarbamoyladenosine (t A), 2-methylthio-N -isopentenyl-ade-nosine (ms i A-37), or wybutosine (yW). yW and its derivatives occur at position 37 in archaeal and eukaryotic phenylalanine tRNA (tRNAphe). The modifications serve to maintain the correct translational reading frame via hydrophobic interactions, which reinforce codon—anticodon pairing and prevent incorrect Watson—Crick base-pairing. Studies have shown that unmodified tRNA leads to translational defects that have been implicated in different pathological states. ... 

As mentioned above, ms i A is one of the modifications observed at position 37 in tRNA molecules. Unlike yW synthesis, the formation of ms i A involves only two enzymes. In the first reaction catalyzed by MiaA, an isopentenyl group is added to the N position of adenosine, yielding the product N -isopentenyl-adenosine... 

In addition to free cytokinins, cytokinin moieties also occur as constituents of some tRNA species of a wide range of organisms including plants [57]. They are located at the strategic 37 position adjacent to the 3 -end of the anticodon [58]. In contrast to the formation of free cytokinins the biosynthetic pathways of tRNA cytokinins are well understood. The first step in their formation is post-transcriptional isopentenylation of Ade using iPP and unmodified tRNA as substrates. This reaction is catalysed by A -isopentenylpyrophosphate tRNA-A -isopentenyltransferase (EC 2.5.1.8) which was partially purified from yeast [59], E. coli [60] and com [61]. This enzyme is encoded by... 

The enzyme A -isopentenyl pyrophosphate tRNA A -isopentenyl transferase has been further characterized. It has a molecular weight of about 55 000, needs dimethylallyl pyrophosphate (3), and is highly stereospecific in its action. The tertiary structure of the tRNA is necessary before a reaction will occur, and the enzyme then modifies the adenosine unit adjacent to the 3 -end of the anticodon. 

C,6H23N504S 381.455 Modified nucleoside found in tRNA s. S-Me, FC-(3-metkyl-2-butenyl) N-f3-Methyl-2-butenyl)-2-(methylthio )adeno-sine, 9CI. FC-Isopentenyl-2-(methylthio) adenosine [20859-00-1]... 

Hall, R.H. N (zl -isopentenyl) adenosine Chemical reactions, biosyn- thesis, metabolism and significance to the structure and function of tRNA. Prog. Nucleic Acid Res. Mol. Biol. 10, 57-86 (1970)... 

MV A would appear to be a better precursor than adenine and its derivatives for studies of cytokinin biosynthesis because of its reduced conversion into undesirable basic compounds [15], and the fact that its incorporation into isopentenyl groups of tRNA cytokinins has been established [15]. However, there are only a few reports of MVA s incorporation into free cytokinins [2, 5], and unfortunately the incorporation was extremely low, preventing proper characterization of the labelled cytokinins. Furthermore, MVA incorporation into cytokinins by V. rosea crown gall tissues could not be detected under conditions which result in maximal incorporation of [ C]-adenine into cytokinins [19]. The uptake of MVA (a mixture of optical isomers) by cell cultures is often very poor. Furthermore the radioactivity taken up is possibly swamped by a large endogenous pool of MVA. This severely limits the chance of detecting MVA s incorporation into cytokinins. 

Bios5mthetic pathways of naturally occurring cytokinins are illustrated in Fig. 29.5. The first step of cytokinin biosynthesis is the formation of A -(A -isopentenyl) adenine nucleotides catalyzed by adenylate isopentenyltransferase (EC 2.5.1.27). In higher plants, A -(A -isopentenyl)adenine riboside 5 -triphosphate or A -(A -isopentenyl)adenine riboside 5 -diphosphate are formed preferentially. In Arabidopsis, A -(A -isopentenyl)adenine nucleotides are converted into fraws-zeatin nucleotides by cytochrome P450 monooxygenases. Bioactive cytokinins are base forms. Cytokinin nucleotides are converted to nucleobases by 5 -nucleotidase and nucleosidase as shown in the conventional purine nucleotide catabolism pathway. However, a novel enzyme, cytokinin nucleoside 5 -monophosphate phosphoribo-hydrolase, named LOG, has recently been identified. Therefore, it is likely that at least two pathways convert inactive nucleotide forms of cytokinin to the active freebase forms that occur in plants [27, 42]. The reverse reactions, the conversion of the active to inactive structures, seem to be catalyzed by adenine phosphoiibosyl-transferase [43] and/or adenosine kinase [44]. In addition, biosynthesis of c/s-zeatin from tRNAs in plants has been demonstrated using Arabidopsis mutants with defective tRNA isopentenyltransferases [45]. 

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