Wheat Embryo Ribonucleates. V. Generation of N2-Dimethylgnanylate When 'Fully Sequenced' Homogeneous Species of Transfer RNA are Used as Substrates for Wheat Embryo Methyltransferases

1975 ◽  
Vol 53 (6) ◽  
pp. 690-697 ◽  
Author(s):  
T. C. Kwong ◽  
B. G. Lane
Keyword(s):  
Molecular Basis ◽  
Transfer Rna ◽  
Target Site ◽  
Common Site ◽  
Tertiary Structures ◽  
Wheat Embryo ◽  
E Coli ◽  
Yeast Trna ◽  
The Subject ◽  
Random Distributions

1. When S-adenosyl[methyl-14C]methiomne and various species of transfer RNA are used as substrates for wheat embryo methyltransferases, the principal site of guanylate-N2 methylation can be shown to be a G-residue between the stems of the dihydrouridine and anticodon loops. This common site of guanylate-N2 methylation is referred to as the interstem target site.2. When the interstem target site is the non-terminal G-residue in a G-C-G-C sequence, as in the cases of Escherichia coli tRNA1Leu, tRNAIle, and tRNA3Ser, there is preponderant dimethylation to yield N2-dimethylguanylate.3. When the interstem target site is part of a U-C-G-U sequence, as in the case of E. coli tRNAtMet, there is diminished dimethylation and correspondingly increased monomethylation to yield N2-monomethylguanylate.4. When the interstem target site is the non-terminal G-residue in an A-U-G-G sequence, as in the case of yeast tRNAAsp, there is negligible dimethylation and almost exclusive monomethylation to yield N2-monomethylguanylate.5. The concerted way in which the primary, secondary, and tertiary structures of tRNA molecules might influence the efficacy of these methylations is the subject of a brief discussion. Attention is also focused on the evolutionary and molecular basis for the generally non-random distributions of methylated oligonucleotide sequences in ribosomal and transfer ribonucleates.

In vitro O2′-methylation of sugars in E. coli RNA. III. Methylation of E. coli transfer RNA by heterologous methylases in particulate-free extracts of wheat embryo

1969 ◽  
Vol 47 (9) ◽  
pp. 863-869
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Transfer Rna ◽  
The Other ◽  
Wheat Embryo ◽  
E Coli ◽  
Pyrimidine Nucleosides ◽  
Other Hand

(1) Transfer ribonucleates (tRNA) from a relaxed strain of E. coli have been methylated by homologous enzymes from E. coli and by heterologous enzymes from wheat embryo, using particulate-free extracts supplemented with [methyl-14C]-S-adenosylmethionine.(2) For both homologous and heterologous systems, the methylation of bases greatly exceeded the methylation of sugars, although the patterns of base and sugar methylation were radically different for homologous and heterologous methylations. Thus, the primary targets for homologous methylation were pyrimidine nucleosides: ribothymidine, the primary product of base methylation, and O2′-methylcytidine in the sequence CmpU, the primary product of sugar methylation. On the other hand, the primary targets for heterologous methylation were purine nucleosides: N2,N2-dimethylguanosine, the primary product of base methylation, and O2′-methylguanosine in the sequence GmpG, the primary product of sugar methylation.(3) The results of an allied study of the methylation of wheat embryo tRNA by homologous wheat embryo methylases and heterologous E. coli methylases have also been reported.

Selective Labelling of the Methyl Carboxylate Substituents Found in the Anticodon Sequences of Some Species of Yeast Transfer RNA

1975 ◽  
Vol 53 (1) ◽  
pp. 1-10 ◽  
Author(s):  
T. D. Kennedy ◽  
B. G. Lane
Keyword(s):  
Methyl Ester ◽  
Room Temperature ◽  
Methyl Esters ◽  
Transfer Rna ◽  
Wheat Embryo ◽  
Yeast Trna ◽  
Selective Labelling

(1) By incubation in 0.1 M NaOH for 10 min at room temperature, it is possible to "saponify" some of the methyl carboxylate linkages in bulk yeast tRNA. By incubation with S-adenosyl[Me-14C]methionine and either homologous (yeast) or heterologous (wheat-embryo) enzymes, it is then possible to "re-esterify" the "saponified" tRNA and thereby effect selective labelling at 5-carboxymethyluridine [Me-14C]methyl ester residues. (2) There is also selective labelling at 2-thio-5-carboxymethyluridine [Me-14C]methyl ester residues when "saponified" yeast tRNA is incubated with S-adenosyl[Me-14C]methionine and homologous (but not heterologous) enzymes. (3) When selectively labelled yeast tRNA is hydrolyzed by RNase T1, both 5-carboxymethyluridine [Me-14C]methyl ester and its 2-thio-analogue are released as part of large oligonucleotides, each of which contains roughly 10 nucleotide residues. (4) There are at least three, and possibly four [Me-14C]-methyl ester-containing oligonucleotides released by RNase T1 digestion of selectively labelled "saponified" yeast tRNA. A comparison of the chromatographic properties of the different [Me-14C]oligonucleotides suggests that the same 5-carboxymethyluridine residues are probably targets for both homologous and heterologous enzymes. (5) The properties of the selectively labelled oligonucleotides are consistent with the view that some of them probably are derived from yeast tRNA3Glu, tRNA2Lys, and tRNA3Arg, ail of which are known to contain 5-carboxymethyl methyl esters as part of their anticodon sequences.

Modified 5′-nucleotides resistant to 5′-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate and N2,N2-dimethylguanosine 5′-phosphate from snake venom hydrolysates of transfer RNA

1976 ◽  
Vol 54 (5) ◽  
pp. 413-422 ◽  
Author(s):  
M. W. Gray
Keyword(s):  
Ribosomal Rna ◽  
Quantitative Measurement ◽  
Chinese Hamster ◽  
Assay Method ◽  
Wheat Embryo ◽  
E Coli ◽  
Yeast Trna ◽  
The Stability ◽  
Hydrolysis Conditions ◽  
Hydrolysis Of

A procedure for the quantitative measurement of the O2′-methylnucleoside constituents of RNA has recently been developed in this laboratory (Gray, M. W. Can. J. Biochem. 53,735–746 (1975)). This assay method is based on the resistance of O2′-methylnucleoside 5′-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5′-nucleotidase (EC 3.1.3.5). In the present investigation, two base-modified 5′-nucleotides, each displaying an unusual resistance to 5′-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2,N2-dimethylguanosine 5′-phosphate [Formula: see text] and 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate (p4abu3U). Because of their resistance to 5′-nucleotidase, [Formula: see text] and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA.Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean ± SD) of p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been determined to be 0.35 ± 0.03 (n = 5) and 0.14 ± 0.02 (n = 4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of [Formula: see text] from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistant to 5′-nucleotidase. The complete absence of[Formula: see text] in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2,N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5′-nucleotidase is a necessary but not sufficient criterion for concluding that a 5′-nucleotide is O2′-methylated.When applied to wheat embryo ribosomal RNA, the analytical methods described in this report failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells (Saponara, A. G. &Enger, M. D. Biochim. Biophys. Acta 349,61–77 (1974)), may not be present in wheat embryo ribosomal RNA.

scholarly journals Public-good driven release of heterogeneous resources leads to genotypic diversification of an isogenic yeast population in melibiose.

2021 ◽  
Author(s):  
Anjali Mahilkar ◽  
Phaniendra Alugoju ◽  
Vijendra Kavatalkar ◽  
Rajeshkannan E. ◽  
Jayadeva Bhat ◽  
...  
Keyword(s):  
Carbon Source ◽  
Public Good ◽  
Molecular Basis ◽  
Model System ◽  
Microbial Populations ◽  
Adaptive Diversification ◽  
E Coli ◽  
Hydrolysis Of ◽  
Convenient Model ◽  
Heterogeneous Resources

Adaptive diversification of an isogenic population, and its molecular basis has been a subject of a number of studies in the last few years. Microbial populations offer a relatively convenient model system to study this question. In this context, an isogenic population of bacteria (E. coli, B. subtilis, and Pseudomonas) has been shown to lead to genetic diversification in the population, when propagated for a number of generations. This diversification is known to occur when the individuals in the population have access to two or more resources/environments, which are separated either temporally or spatially. Here, we report adaptive diversification in an isogenic population of yeast, S. cerevisiae, when propagated in an environment containing melibiose as the carbon source. The diversification is driven due to a public good, enzyme α-galactosidase, leading to hydrolysis of melibiose into two distinct resources, glucose and galactose. The diversification is driven by a mutations at a single locus, in the GAL3 gene in the GAL/MEL regulon in the yeast.

ORGANISATION AND EXPRESSION OF THE E. COLI PHENYLALANYL-TRANSFER RNA SYNTHETASE OPERON

1982 ◽  
pp. 25-47 ◽  
Author(s):  
MATHIAS SPRINGER ◽  
JACQUELINE PLUMBRIDGE ◽  
MARIE TRUDEL ◽  
MARIANNE GRUNBERG-MANAGO ◽  
GUY FAYAT ◽  
...  
Keyword(s):  
Transfer Rna ◽  
E Coli

scholarly journals Molecular basis of abasic site sensing in single-stranded DNA by the SRAP domain of E. coli yedK

2019 ◽  
Vol 47 (19) ◽  
pp. 10388-10399 ◽  
Author(s):  
Na Wang ◽  
Hongyu Bao ◽  
Liu Chen ◽  
Yanhong Liu ◽  
Yue Li ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Substrate Binding ◽  
Specific Substrate ◽  
Abasic Site ◽  
E Coli ◽  
Abasic Sites ◽  
Ap Sites ◽  
Ap Site

Abstract HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.

scholarly journals The effect of hypermethylation on the functional properties of transfer ribonucleic acid. Formation of aminoacyl-transfer-ribonucleic acid

1969 ◽  
Vol 114 (2) ◽  
pp. 429-435 ◽  
Author(s):  
David J. Pillinger ◽  
John Hay ◽  
Ernest Borek
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
Control Sample ◽  
Transfer Rna ◽  
E Coli ◽  
Functional Sites ◽  
The Individual ◽  
Almost All ◽  
Aminoacyl Transfer ◽  
Kinetics Of

1. The ability of chemically hypermethylated Escherichia coli B transfer RNA to accept 19 amino acids was studied and the results were compared with those obtained with a control sample of E. coli B transfer RNA incubated under similar conditions in the absence of methylating agent. 2. There is a marked decrease in the ability of the modified transfer RNA to accept amino acids in almost all instances. 3. The acceptance of cysteine appears to be unique in that it is enhanced in the hypermethylated transfer RNA. 4. More detailed studies on the kinetics of acceptance for six amino acids is presented, emphasizing the variation in response of the individual amino acids. 5. Increasing hypermethylation causes a progressive decrease in the amino acid acceptance. 6. The results are discussed in terms of methylation at functional sites within the transfer RNA and possible conformational alterations to the structure of the macromolecule.

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