Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase
A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNA/sup fMet/). Incubation of the affinity-labeling tRNA/sup fMet/ derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNA/sup fMet/, but not by noncognate tRNA/sup Phe/. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.
- Research Organization:
- Albert Einstein College of Medicine, Bronx, NY
- OSTI ID:
- 5515578
- Journal Information:
- Biochemistry; (United States), Vol. 26:22
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
LABELLED COMPOUNDS
CROSS-LINKING
LIGASES
TRANSFER RNA
AMINO ACID SEQUENCE
CARBON 14 COMPOUNDS
ESCHERICHIA COLI
ION EXCHANGE CHROMATOGRAPHY
LYSINE
MOLECULAR STRUCTURE
SULFUR 35
URIDINE
AMINO ACIDS
AZINES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
EVEN-ODD NUCLEI
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
POLYMERIZATION
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
RNA
SEPARATION PROCESSES
SULFUR ISOTOPES
URACILS
550201* - Biochemistry- Tracer Techniques