Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “tRNA modification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources
PubMed1985

Eukaryotic RNA polymerases.

This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.

Animals↗
PubMed2005

Hypomodification of transfer RNA in cancer with respect to queuosine.

Queuosine is a highly modified nucleoside analogue of guanosine. It is present only in the first position of anticodon loop of specific tRNA i.e., tRNA(his), tRNA(asp), tRNA(asn) and tRNA(tyr) and post transcriptionally modified with base-for-base exchange of guanine to queuine. The transfer RNA modifying enzyme transfer RNA guanine transglycosylase (TGTase) catalyzes the modification of tRNAs. Transfer RNA is completely modified with respect to queuosine in mature tissue, however modification is often incomplete in mitotically active cells. Hypomodification of transfer RNA is correlated with cell proliferation and malignancy. In the present study queuosine modification of transfer RNA and TGTase activity is compared in normal, Dalton's lymphoma ascites transplanted (DLAT) cancerous and queuine treated DLAT cancerous mouse liver. Transfer RNA of cancerous mouse is hypomodified in terms of queuosine modification. TGTase activity of cancerous mouse is found to decrease to less then half of enzyme activity of normal mouse; suggesting that the enzyme may be responsible for transfer RNA hypomodification. Exogenous treatment of queuine during development of cancer improves the queuosine modification of transfer RNA. The activators NaPP and ATP enhance TGTase activity of normal and DLAT cancerous mouse, where as 7mG inhibits the TGTase activity.

Animals↗
PubMed1986

[Determination of phosphate residues participating in the formation of the spatial structure of tRNA- Leu IAG from cow mammary glands].

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.

Animals↗
PubMedDate not supplied

[Affinity modification of Escherichia coli ribosomes with photoactivated analogs of mRNA].

Oligoribonucleotide derivatives containing the photoactivated arylazidogroup at 5'-end of the oligonucleotide fragment [2-(N-2,4-dinitro-5-azidophenyl) aminoethyl] phosphamides of the oligoribonucleotides, azido-NH (CH2)2NHpN (pN) n-1, were prepared. It was demonstrated that azido-NH(CH2)2NHpA(pA)4 and azido-NH (CH2)2NHpU (pU)3 stimulate the binding of the codonspecific aminoacyl-tRNA with ribosome. After irradiation of the ternary complex ribosome-azido-NH (CH2)2NHpU (pU) n-1 X tRNA with UV-light (lambda greater than 350 nm) covalent binding of the reagent to ribosome occurs. Up to 10% of the reagent, bound in the ternary complex with ribosome, is cross-linked with the ribosomal proteins of 30S and 50S subunits. The ribosomal RNA are not modified by azido-NH (CH2)2NHpU (pU) n-1. The proteins of 30S and 50S subunits, modified with azido-NH (CH2)2NHpU (pU) n-1 with n = 4,7 and 8, were identified. It is shown that proteins of 30S subunits S3, S4, S9, S11, S12, S14, S17, S19, S20 undergo modification. The proteins of 50S subunits L2, L13, L16, L27, L32, L33 are modified. The set of the modified proteins essentially depends on the length of the oligonucleotide part of the reagent and on occupancy of ribosome A-site by a molecule of tRNA.

Chromatography, Affinity↗
PubMed1994

Methionyl-tRNA synthetase needs an intact and mobile 332KMSKS336 motif in catalysis of methionyl adenylate formation.

The family of aminoacyl-tRNA synthetases may be split into two classes according to the occurrence of specific combinations of peptide motifs. This study deals with the functional role of the KMSKS motif, which, in association with the HIGH motif, defines class 1 aminoacyl-tRNA synthetases. Each residue in the 332KMSKS336 sequence of Escherichia coli methionyl-tRNA synthetase, as well as R337 and the two surrounding G330 and G338 residues, were mutagenized. The comparison of the kinetic and equilibrium parameters of the methionine activation reaction sustained by the resulting variants enables the following conclusions to be drawn. (1) Mutation of all the residues studied strongly destabilizes the transition state for the formation of methionyl adenylate whilst changing moderately the stability of the ground state ternary complex enzyme, methionine: ATP-Mg2+. The consequences of the mutations are also reflected at the level of the stability of the ground state enzyme, methionyl adenylate:PPi-Mg2+ complex which is systematically decreased. (2) The substitution with alanine of any one of the three basic residues K332, K335 and R337 destabilizes the transition state by more than 3.2 kcal/mol, while substitution of the non-basic residues M333, S334 or S336 destabilizes it by at most 2.5 kcal/mol. Such a difference may reflect different modes of action of the residues, with the basic ones directly interacting with the beta and gamma phosphates of the ATP-Mg2+ substrate and the non-basic ones playing a structural role and/or participating in mobility of the enzyme region carrying the motif. (3) Modification of G330 or G338 to a proline markedly decreases the kinetic rate of methionyl adenylate formation. This behaviour suggests that the flexibility of the KMSKS loop in the structure of methionyl-tRNA synthetase is required to reach the transition state during formation of methionyl adenylate.

Adenosine↗
PubMed2006

Ribonucleome analysis identified enzyme genes responsible for wybutosine synthesis.

Wybutosine (yW) is a hyper modified guanosine with a tricyclic base found at the 3'-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports reading frame maintenance by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). We found four new genes YPL207w, YML005w, YOL141w and YGL050w (named TYW1, TYW2, TYW3 and TYW4, respectively) to be responsible for wybutosine (yW) synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. Multi-step enzymatic formation of yW was partially reconstituted in vitro using recombinant proteins. The results suggested that yW synthesis might proceed through sequential reactions in a complex formed by multiple protein components assembled with the precursor tRNA.

Gene Deletion↗
PubMedDate not supplied

The primary structures of three yeast mitochondrial serine tRNA isoacceptors.

Yeast mitochondria contain several isoaccepting species of serine-tRNA. The relative amount of these isoacceptors varies according to the conditions used to grow the yeast cells. In order to gain insight into the structural differences among these isoacceptors, the three mitochondrial tRNAsSer, which are present in derepressed yeast cells, have been sequenced. The primary structure of tRNASer1 differs considerably from that of tRNASer2; these two isoacceptors have only 39 nucleotides in common. In contrast, tRNASer3 differs from tRNASer2 by only one post-transcriptional modification: the psi residue in position 28 of tRNASer2 is replaced by a normal U in tRNASer3. Unlike tRNASer2 and tRNASer3, the primary sequence of tRNASer1 shows two unusual structural features: it has a D in position 14 instead of the "universal" A14 of the standard tRNA cloverleaf and it contains two G residues between the D-stem and the anticodon-stem. Considering their respective anticodons, tRNASer1 should recognize the two serine codons A-G-C and A-G-U, whereas both tRNASer2 and tRNASer3 should recognize all four serine codons of the U-C-N series.

Amino Acyl-tRNA Synthetases↗
PubMed2003

A new strategy for the site-specific modification of proteins in vivo.

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed1985

Euglena gracilis chloroplast elongation factor Tu. Interaction with guanine nucleotides and aminoacyl-tRNA.

The interaction of the chloroplast elongation factor Tu (EF-Tuchl) from Euglena gracilis with guanine nucleotides and aminoacyl-tRNA has been investigated. The apparent dissociation constant at 37 degrees C for the EF-Tuchl X GDP complex is about 3 X 10(-7) M and for the EF-Tuchl X GTP complex, it is about 1 order of magnitude higher. The sulfhydryl modifying reagent N-ethylmaleimide severely inhibits the polymerization activity of Euglena EF-Tuchl. In the presence of N-ethylmaleimide, the dissociation constant for the modified EF-Tuchl X GDP complex is increased by an order of magnitude. Conversely, both GDP and GTP protect EF-Tuchl from the modification. The polymerization activity of EF-Tuchl is also sensitive to the antibiotic kirromycin. In the presence of kirromycin, the apparent dissociation constant for the EF-Tuchl X GTP complex is lowered 10-fold. The interaction of aminoacyl-tRNA with EF-Tuchl was investigated by examining the ability of EF-Tuchl to prevent the spontaneous hydrolysis of Phe-tRNA and by gel filtration chromatography. The binding of aminoacyl-tRNA to EF-Tuchl occurs only in the presence of GTP indicating the formation of the ternary complex EF-Tuchl X GTP X Phe-tRNA. The effect of kirromycin on the interaction was also investigated. In the presence of kirromycin, no interaction between EF-Tuchl and Phe-tRNA is observed, even in the presence of GTP.

Amino Acid Sequence↗
PubMed1994

A cysteine in the C-terminal region of alanyl-tRNA synthetase is important for aminoacylation activity.

Alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a multimeric enzyme that catalyzes the esterification of alanine to tRNA(Ala) in the ATP-dependent aminoacylation reaction. The functional binding of all three substrates follows Michaelis-Menten kinetics. The role of cysteines in this enzyme has been evaluated via modification of these residues with p-(hydroxymercuri)phenylsulfonic acid, monobromobimane, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The former two reagents induce nearly complete inactivation of AlaRS aminoacylation activity and the release of all tightly bound zinc. In the case of mild DTNB treatment, only two of the six cysteines in AlaRS are modified, with release of all zinc and partial loss of aminoacylation activity. These experiments indicate the importance of one or more cysteines, other than those thought to be coordinated with zinc, in the aminoacylation reaction. Substitution of each of the cysteine residues outside the zinc-binding motif with serine does not disrupt zinc binding. However, the cysteine most removed in primary sequence from the active site (Cys665) is identified as important in the aminoacylation step. Mutation of Cys665 to serine induces a 120-fold decrease in the catalytic efficiency of this enzyme, primarily through a kcat effect, and introduces sigmoidal kinetics (nH = 1.8) with respect to the RNA substrate. The results demonstrate that a simple manipulation in the C-terminal region can introduce positive cooperativity in this otherwise noncooperative enzyme.

Acylation↗
PubMed1986

Functional modification of rat liver ribosomes by the in vitro action of N-methyl-N'-nitro-N-nitrosoguanidine.

The mechanism by which N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inhibits protein synthesis has been studied in a rat liver cell free system. Using preformed aminoacyl-tRNA it was observed that incorporation of amino acid into polyribosomal protein was inhibited in the presence of low concentration of MNNG. This inhibition was not reversed by increasing the concentration of soluble factors. Transfer RNAs modified previously by treatment with MNNG and subsequently esterified with amino acids were transferred to polyribosomes with the same efficiency as those species which were not modified. Polyribosomes, on the other hand, lost activity to incorporate amino acids after pretreatment with MNNG. This inactivation was dependent on the concentration of MNNG with which polyribosomes were treated. When poly(U) was used with MNNG-treated polyribosomes, its translation, after correction for endogenous translation, was also found to be significantly low as compared to the case with untreated polyribosomes. Purified ribosomes stripped of endogenous mRNA when treated with increasing concentrations of MNNG progressively lost ability to support polyphenylalanine synthesis programmed by poly(U). The treated ribosomes, however, neither inhibited the activity of control ribosomes nor induced any loss of fidelity of translation by poly(U). It is concluded that MNNG inhibits protein synthesis through functional inactivation of ribosomes resulting from direct modification of ribosomal proteins possibly involving nitroguanidination of lysine residues.

Animals↗
PubMed2003

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): deciphering the basis for nucleotide selection.

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N6-methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2'-O-methyladenosine, 2'-deoxy-2'-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3'-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.

Base Sequence↗
PubMed2006

The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation.

Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.

Archaea↗
PubMed1988

Incorporation of single dinitrophenyl-modified proteins into the 30 S subunit of Escherichia coli ribosomes by total reconstitution.

In this first of two consecutive papers, the main objective of which is to present a new approach to the systematic localization of individual proteins located in the Escherichia coli ribosome by immunoelectron microscopy, we describe the derivatization of several purified 30 S proteins (S12, S21, S14, S19, S18, S17) with 2,4-[3,5-3H]dinitrofluorobenzene at pH 7.4 and 8.4 and the uptake of each dinitrophenylated protein in place of the corresponding unmodified protein into totally reconstituted 30 S subunits. Reverse-phase high performance liquid chromatography is used to purify the proteins, to separate and characterize the products of 2,4-[3,5-3H]dinitrofluorobenzene modification, and to analyze the protein composition of the reconstituted subunits. The extent of dinitrophenyl (DNP) modification is estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214 and 360 nm. DNP derivatives of each of the six proteins are efficiently incorporated into reconstituting 30 S subunits. Incorporation of any of the six DNP-modified proteins does not interfere with binding of Phe-tRNA(Phe) in a poly(U)-dependent manner. This result, as well as data showing that unmodified protein competes with DNP-protein for uptake during reconstitution, provide evidence that each DNP-protein occupies the same position in 30 S subunit as does unmodified protein. In general, for a given protein, unmodified and/or less modified forms are incorporated in preference to more modified forms. Modification of protein S19 at pH 7.4 proceeds with selective formation of one derivative in high yield. Reverse-phase high performance liquid chromatography analysis of acid hydrolysates of a purified sample of this derivative, as well as of peptides derived from it by digestion with Staphylococcus aureus protease, show the N-terminal proline to be the predominant site of DNP-derivatization.

Amino Acids↗
PubMed1982

The nucleotide sequence of tRNA4Val of Drosophila melanogaster. Chloroacetaldehyde modification as an aid to RNA sequencing.

The nucleotide sequence of tRNA4Val from Drosophila melanogaster was determined to be pGUUUmCCGUm1GGUG psi AGCGGDU(acp3U)AUCACA psi CUGCCmUIACAm5CGCAGAAGm7GCCCCCGGT psi CGm1AUCCCGGGCGGAAACACCA. It is probable that residue C 49 is modified to m5C. The use of tRNA modified with chloroacetaldehyde to overcome secondary structure problems in sequencing is described.

Acetaldehyde↗