Characterization of bacteriophage T4 regA protein-nucleic acid interactions.
The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.
Webster
K R
KR
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06511.
Spicer
E K
EK
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005813:Genes, Synthetic; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D010442:Peptide Chain Initiation, Translational; D011131:Polyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D013604:T-Phages / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D015345:Oligonucleotide Probes; D011131:Polyribonucleotides; D012367:RNA, Viral; C066700:RegA protein, Enterobacteria phage T4; D012097:Repressor Proteins; D014764:Viral Proteins