Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.
The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.
Webster
K R
KR
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06511.
Keill
S
S
Konigsberg
W
W
Williams
K R
KR
Spicer
E K
EK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D009843:Oligoribonucleotides; D010446:Peptide Fragments; D012333:RNA, Messenger; D012367:RNA, Viral; C072722:RegA protein, Bacteria; D014357:Trypsin