Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.     

The CDK5 repressor CDK5RAP1 is a methylthiotransferase acting on nuclear and mitochondrial RNA

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.     

ms2i6A deficiency enhances proofreading in translation.

The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine. Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro. Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged. Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP. ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type. ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).     

N6-isopentenyladenosine a new potential anti-angiogenic compound that targets human microvascular endothelial cells in vitro

Abstract

N6-isopentenyladenosine is an anti-proliferative and pro-apoptotic atypical nucleoside for normal and tumor cells. Considering the role of angiogenesis in various diseases, we investigated the cytotoxic effect of N6-isopentenyladenosine on human microvascular endothelial cells, protagonists in angiogenesis. Our results show that N6-isopentenyladenosine induced a significant reduction of cell viability, upregulated p21 and promoted caspase-3 cleavage in a dose dependent manner leading to apoptotic cell death as detected by FACS analysis. To understand structure-function relationship of N6-isopentenyladenosine, we investigated the effect of some N6-isopentenyladenosine analogs. Our results suggest that N6-isopentenyladenosine and some of its derivatives are potentially novel angiostatic agents and might be associated with other anti-angiogenic compounds for a better outcome.     

The rate of peptidyl-tRNA dissociation from the ribosome during minigene expression depends on the nature of the last decoding interaction

The expression of some very short open reading frames (ORFs) in Escherichia coli results in peptidyl-tRNA accumulation that is lethal to cells defective in peptidyl-tRNA hydrolase activity. In an attempt to understand the factors that affect this phenotype, we have surveyed the toxicity of a complete set of two-codon ORFs cloned as minigenes in inducible expression vectors. The minigenes were tested in hydrolase-defective hosts and classified according to their degree of toxicity. In general, minigenes harboring codons belonging to the same box in the standard table of the genetic code mediated similar degrees of toxicity. Moreover, the levels of peptidyl-tRNA accumulation for synonymous minigenes decoded by the same tRNA were comparable. However, two exceptions were observed: (i) expression of minigenes harboring the Arg codons CGA, CGU, and CGC, resulted in the accumulation of different levels of the unique peptidyl-tRNAArg-2 and (ii) the toxicity of minigenes containing CUG and UCU codons, each recognized by two different tRNAs, depended on peptidyl-tRNA accumulation of only one of them. Non-toxic, or partly toxic, minigenes prompted higher accumulation levels of peptidyl-tRNA upon deprivation of active RF1, implying that translation termination occurred efficiently. Our data indicate that the nature of the last decoding tRNA is crucial in the rate of peptidyl-tRNA release from the ribosome.     

Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.     

Yeast Pth2 is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway

Ubiquitin-like (UBL)-ubiquitin-associated (UBA) proteins such as Rad23 and Dsk2 mediate the delivery of polyubiquitinated proteins to the proteasome in the ubiquitin-proteasome pathway. We show here that budding yeast peptidyl-tRNA hydrolase 2 (Pth2), which was previously recognized as a peptidyl-tRNA hydrolase, is a UBL domain-binding protein that participates in the ubiquitin-proteasome pathway. Pth2 bound to the UBL domain of both Rad23 and Dsk2. Pth2 also interacted with polyubiquitinated proteins through the UBA domains of Rad23 and Dsk2. Pth2 overexpression caused an accumulation of polyubiquitinated proteins and inhibited the growth of yeast. Ubiquitin-dependent degradation was accelerated in the pth2Delta mutant and was retarded by overexpression of Pth2. Pth2 inhibited the interaction of Rad23 and Dsk2 with the polyubiquitin receptors Rpn1 and Rpn10 on the proteasome. Furthermore, Pth2 function involving UBL-UBA proteins was independent of its peptidyl-tRNA hydrolase activity. These results suggest that Pth2 negatively regulates the UBL-UBA protein-mediated shuttling pathway in the ubiquitin-proteasome system.     

Dissociation of peptidyl-tRNA from ribosomes is perturbed by streptomycin and by strA mutations

Summary Peptidyl-tRNA may dissociate preferentially from ribosomes during protein synthesis when it is inappropriate to, does not correctly complement, the messenger RNA. To test this idea, growing cultures of Escherichia coli were treated with streptomycin to increase the frequency of errors during protein synthesis. Since the treated cells had a temperature-sensitive peptidyl-tRNA hydrolase and could not destroy dissociated peptidyl-tRNA, it was possible to measure the rate of its accumulation after raising the temperature to non-permissive conditions. Both low and high doses of streptomycin enhanced the rate of dissociation and accumulation of peptidyl-tRNA. The rank order of rates of dissociation/accumulation of various isoaccepting tRNA families was not significantly altered by the drug treatment. We concluded that streptomycin stimulated a normal pathway for dissociation of peptidyl-tRNA. Two streptomycin-resistent strains of E. coli had higher rates of dissociation of peptidyl-tRNA than did their sensitive parent strain. When treated with high doses of the drug, the resistant strains showed slightly reduced rates of dissociation of peptidyl-tRNA. These results were interpreted in terms of a two state, two site model for protein synthesis: streptomycin enhances the binding of aminoacyl-tRNA to a tight state of the ribosome A site; the strA mutation enhances translocation to a loose state of the ribosome P site.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.     

Accumulation of peptidyl tRNA is lethal to Escherichia coli.

A mutant strain of Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase grows at 30 degrees C but, when shifted to 40 degrees C, dies at rates affected by physiological, pharmacological, and genetical perturbations. The rate of killing correlates with the relative accumulation of peptidyl-tRNA, suggesting that it is responsible for the death of the cells.     

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3-8 codon minigenes containing AGAAGA codons before the stop codon. Beta-galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, beta-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 microg/microl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of beta-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNA(Arg4). These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.     

The 2'-OH group of the peptidyl-tRNA stabilizes an active conformation of the ribosomal PTC

The ribosome accelerates the rate of peptidyl transfer by >10(6)-fold relative to the background rate. A widely accepted model for this rate enhancement invokes entropic effects whereby the ribosome and the 2'-OH of the peptidyl-tRNA substrate precisely position the reactive moieties through an extensive network of hydrogen bonds that allows proton movement through them. Some studies, however, have called this model into question because they find the 2'-OH of the peptidyl-tRNA to be dispensable for catalysis. Here, we use an in vitro reconstituted translation system to resolve these discrepancies. We find that catalysis is at least 100-fold slower with the dA76-substituted peptidyl-tRNA substrate and that the peptidyl transferase centre undergoes a slow inactivation when the peptidyl-tRNA lacks the 2'-OH group. Additionally, the 2'-OH group was found to be critical for EFTu binding and peptide release. These findings reconcile the conflict in the literature, and support a model where interactions between active site residues and the 2'-OH of A76 of the peptidyl-tRNA are pivotal in orienting substrates in this active site for optimal catalysis.     

Presence of 2-methylthioribosyl-trans-zeatin in Azotobacter vinelandii tRNA.

Hydroxylated cytokinin, 2-methylthio-N6-(4-hydroxy-3-methylbut-2-enyl) adenosine, was found in the tRNA of Azotobacter vinelandii. This cytokinin had the trans configuration, unlike the cis configuration reported for that from other bacteria. Culture-condition-dependent changes in the content of this thiocytokinin and a few other thionucleosides in the tRNA of this bacterium have been observed.     

Synthesis and evaluation of in vitro anticancer activity of some novel isopentenyladenosine derivatives

The present study describes the synthesis, the characterization and the evaluation of some derivatives of N⁶-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(3′-deoxy-β-d-ribofuranosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(β-d-ribofuranosyl)-purine, inhibit the growth of T24 cells, although to a lower extent than N⁶-isopentenyladenosine. We conclude that the integrity of ribosidic and purine moiety and the N⁶ position of the chain are essential for maintaining the antiproliferative activity.     

Structural and functional insights into peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.     

Nonenzymic translocation and spontaneous release of noncognate peptidyl transfer ribonucleic acid from Escherichia coli ribosomes

Poly(uridylic acid)-programmed ribosomes have been used to synthesize the noncognate peptidyl-tRNA Ac-Phe-Tyr-tRNATyr and its cognate counterpart Ac(Phe)2-tRNAPhe. After synthesis, Ac(Phe)2-tRNAPhe remains, as expected, in the ribosomal acceptor (A) site, but the noncognate AcPhe-Tyr-tRNATyr does not; part of it spontaneously falls off the ribosome and the rest translocates, without elongation factor (EF) G, to the ribosomal donor site. The inhibitor of translocation viomycin prevents both the spontaneous release and the nonenzymatic translocation by confining the noncognate peptidyl-tRNA to the A site. Under these conditions, the interaction of AcPhe-Tyr-tRNATyr with the A site appears to be similar to that of Ac(Phe)2-tRNAPhe without the antibiotic, and EF-G promotes the translocation and subsequent elongation of both peptidyl-tRNAs to comparable extents. The results indicate that, without viomycin, the noncognate peptidyl-tRNA is weakly held in the ribosomal A site and support the proposal that the release of peptidyl-tRNA occurring during protein synthesis in vivo is related to a ribosomal editing mechanism which discards mistranslated nascent proteins [Menninger, J. R. (1977) Mech. Ageing Dev. 6, 131].     

Conformational preferences of modified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) adenine and 2-methylthio-N6-(N-threonylcarbonyl) adenine by the quantum chemical PCILO calculations

Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) Adenine, m6tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS 2tc6 Ade alike the threonylcarbonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(1) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS 2tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(delta 2-isopentenyl) Adenine, mS 2i6 Ade, where the 2-methylthio group orients trans to the C(2)-N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA^Arg4, CGA is recognized by the abundant tRNA^Arg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNA^Arg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNA^Arg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNA^Arg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth⁺ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome.