Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Substrate specificity for 4-thiouridine modification in Escherichia coli.

Charles T Lauhon1, Whitney M Erwin, Giangthy N Ton

  • 1School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, USA. clauhon@facstaff.wisc.edu

The Journal of Biological Chemistry
|March 24, 2004
PubMed
Summary

The biosynthesis of 4-thiouridine (s4U) in E. coli tRNA requires specific RNA structures, not just sequence, for recognition by ThiI and IscS enzymes. Minimal substrates reveal structural elements crucial for efficient s4U modification.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli.

Nucleic acids research·2021
Same author

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA.

Journal of bacteriology·2019
Same author

Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification.

Nucleic acids research·2014
Same author

Vandetanib: a novel targeted therapy for the treatment of metastatic or locally advanced medullary thyroid cancer.

American journal of health-system pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists·2013
Same author

Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase-RNA complex.

Acta crystallographica. Section F, Structural biology and crystallization communications·2013
Same author

Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE.

Microbial pathogenesis·2013

Area of Science:

  • Molecular Biology
  • RNA Biochemistry
  • Enzymology

Background:

  • 4-thiouridine (s4U) is a modified nucleoside found in bacterial tRNA.
  • Its biosynthesis in E. coli involves the thiamin pathway enzyme ThiI and cysteine desulfurase IscS.
  • Understanding the substrate requirements for s4U modification is key to elucidating its biological role.

Purpose of the Study:

  • To determine the minimal structural requirements for E. coli tRNA substrate recognition by ThiI and IscS for s4U modification.
  • To investigate the role of specific RNA sequences and structural features in the T loop and 3' terminus.
  • To identify factors limiting s4U modification to tRNA in vivo.

Main Methods:

  • Deletion analysis of unmodified E. coli tRNA(Phe) to create minimal RNA substrates.

Related Experiment Videos

  • In vitro assays to measure s4U modification activity.
  • Mutagenesis of the T loop and 3' terminus to assess sequence and structural impacts.

    • Main Results:

    • A mini-helix containing stacked acceptor and T stems with a bulged loop of at least 4 nucleotides, including the target uridine, is the minimal substrate.
    • The T loop primary sequence is not essential for recognition; a tetraloop substitution enhanced activity.
    • The 3' terminus requires a specific distance and an overhang for efficient modification, with blunt termini showing minimal activity.

    Conclusions:

    • s4U modification is primarily dictated by RNA secondary and tertiary structural constraints rather than specific primary sequences outside of U8.
    • A two-piece RNA system (substrate RNA and guide RNA) can be efficiently modified, suggesting flexibility in enzyme-substrate interaction.
    • These structural requirements likely explain the specificity of s4U modification to tRNA molecules in the cellular environment.