Mutagenesis of the COOH-terminal region of bacteriophage T4 regA protein.
The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of > 12 T4 proteins. Earlier studies demonstrated that photocross-linking of the 122-residue regA protein to (dT)16 occurs at two sites, with the major site occurring at Phe-106. Amino acid substitutions were introduced at Phe-106 to evaluate its role in nucleic acid binding. Binding affinities of mutants F106C, F106V, and F106Y for nonspecific and specific RNA ligands indicated little difference between the Kapp of the mutants and wild type regA protein, for either poly(U) or for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does not contribute measurably to the overall free energy of binding. Partial proteolysis of regA protein was carried out to further probe its domain structure. Chymotryptic cleavage produced a fragment of 11,095 Da that has reduced affinity for poly(U) and that contains the first 93 residues of regA protein. Interestingly, proteolysis of regA protein is reduced in the presence of the specific target, gene 44 RNA. Two deletion mutants, 1-->94 and 1-->109, have also been cloned and purified. The binding affinities of these deletion mutants indicated a 100-1000-fold reduction in their affinities for poly(U). These studies indicate the last 13 amino acids in regA protein make a significant contribution to RNA binding.
O'Malley
S M
SM
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Sattar
A K
AK
Williams
K R
KR
Spicer
E K
EK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011072:Poly U / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; C072722:RegA protein, Bacteria; D014157:Transcription Factors; D011072:Poly U; D002918:Chymotrypsin