A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase

Harris, Charles L.; Kolanko, Christopher J.

doi:10.1016/0003-2697(89)90271-6

Cited by 7 publications

(8 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Deletion Analysis of tRNA Phe Transcripts-We used a DEAE filter disc assay described previously (34) to obtain the specific activity for s 4 U modification of RNA variants. The assay was measured for the incorporation of 35 S from L-[ 35 S]cysteine into tRNA that was specifically bound on the discs, and the rates were assessed in the linear range of the progress curve at Ͻ10% of the product formation.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The biosynthesis of 4-thiouridine (s 4 U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from L-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s 4 U. In this work, we show through deletion analysis of unmodified E. coli tRNA Phe that the minimum substrate for s 4 U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the T⌿C primary sequence is not a recognition element. An unmodified tRNA Phe transcript in which the 3 -terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3 terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s 4 U modification is limited in the cell to tRNA.

show abstract

Section: Resultsmentioning

confidence: 99%

“…s 4 U Assays-Assays for s 4 U synthesis were conducted using DEAE filter discs using a modification of a method described previously (20,34). This assay measures the incorporation of 35 4 U formation at 10 and 20 M were the same within error.…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Although both thiI and nuVC E. coli mutants are auxotrophic for thiazole, the ThiI protein does not bind PLP (12). Therefore, it is unclear whether thiI corresponds to the nuVA or nuVC locus.Efforts to further purify these enzymes from wild-type strains in a number of laboratories have not been successful, as the enzymes become unstable during purification (9,14). Previous purification attempts used either bulk E. coli or Saccharomyces cereVisiae tRNA as substrate, the latter of…”

mentioning

confidence: 99%

“…Efforts to further purify these enzymes from wild-type strains in a number of laboratories have not been successful, as the enzymes become unstable during purification (9,14). Previous purification attempts used either bulk E. coli or Saccharomyces cereVisiae tRNA as substrate, the latter of…”

mentioning

confidence: 99%

IscS Is a Sulfurtransferase for the in Vitro Biosynthesis of 4-Thiouridine in Escherichia coli tRNA

1999

View full text Add to dashboard Cite

We have improved the in vitro assay for 4-thiouridine (s(4)U) biosynthesis in Escherichia coli tRNA by substituting an unmodified tRNA transcript as substrate and including recombinant ThiI protein, a known factor required for s(4)U synthesis. Using this assay, we have purified an enzyme from wild-type E. coli that is able to provide sulfur for s(4)U synthesis in vitro. The purified protein has a molecular weight of 45 kDa and contains pyridoxal phosphate as a cofactor. This protein catalyzes sulfur transfer from cysteine to tRNA and is analogous to factor C previously reported (Lipsett, M. N. (1972) J. Biol. Chem. 247, 1458-1461). UV spectroscopy and HPLC analysis of thiolated tRNA and its digests confirm that the product of the in vitro reaction is s(4)U. N-Terminal sequence analysis of the purified protein identifies it as IscS, a recently characterized NifS-like cysteine desulfurase that mobilizes sulfur for the synthesis of [Fe-S] clusters. We have cloned and overexpressed iscS and show that the recombinant protein displayed tRNA sulfurtransferase activity equal to that of the native protein. We also show that, of the multiple proteins in E. coli with cysteine desulfurase activity as observed by native gel staining, only IscS is able to mobilize the sulfur for transfer to tRNA. Our identification of IscS as a tRNA sulfurtransferase provides support for this activity in vivo and further expands the role for NifS proteins as versatile sulfur-carrying enzymes.

show abstract

“…[2][3][4][5][6] DNA, which consists of anions at neutral pH, can be obtained by modulating the salt concentration in the mobile phase or by altering the protein charge through adjusting the mobile phase to pH. [7][8][9][10] However, in those researches, the separation efficiency of the DNA was low. Recently, researchers have found that the efficiency of ion-exchange chromatography can be improved by using small solid carriers.…”

Section: Introductionmentioning

confidence: 99%

A new ion-exchange adsorbent with paramagnetic properties for the separation of genomic DNA

Feng

Luan

Pan

et al. 2011

Analyst

View full text Add to dashboard Cite

A new ion-exchange adsorbent (IEA) derived from Fe(3)O(4)/SiO(2)-GPTMS-DEAE with paramagnetic properties was prepared. Fe(3)O(4) nanoparticles were firstly prepared in water-in-oil microemulsion. The magnetic Fe(3)O(4) particles were modified in situ by hydrolysis and condensation reactions with tetraethoxysilane (TEOS) to form the core-shell Fe(3)O(4)/SiO(2). The modified particles were further treated by 3-glycidoxypropyltrimethoxysilane (GPTMS) to form Fe(3)O(4)/SiO(2)-GPTMS nanoparticles. Fe(3)O(4)/SiO(2)-GPTMS-DEAE nanoparticles (IEA) were finally obtained through the condensation reaction between the Cl of diethylaminoethyl chloride-HCl (DEAE) and the epoxy groups of GPTMS in the Fe(3)O(4)/SiO(2)-GPTMS. The obtained IEA has features of paramagnetic and ion exchange properties because of the Fe(3)O(4) nanoparticles and protonated organic amine in the sample. The intermediates and final product obtained in the synthesis process were characterized. The separation result of genomic DNA from blood indicated that Fe(3)O(4)/SiO(2)-GPTMS-DEAE nanoparticles have outstanding advantages in operation, selectivity, and capacity.

show abstract

A rapid diethylaminoethyl paper disk assay for transfer RNA sulfurtransferase

Cited by 7 publications

References 12 publications

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

IscS Is a Sulfurtransferase for the in Vitro Biosynthesis of 4-Thiouridine in Escherichia coli tRNA

A new ion-exchange adsorbent with paramagnetic properties for the separation of genomic DNA

Contact Info

Product

Resources

About