Abstract
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.
Highlights
The bacteriophage T4 regA protein is a translational repressor that regulates the synthesis of > 12 T4 proteins
We have previously shown that repression of T4 gene 44 by regA protein involves the recognition of a specific RNA element, in which apparently both the sequence and structure are of importance (Webster and Spicer, 1990; Szewczak et al, 1991)
More recent studies have focused on structure-function relationships in regA protein in an effort to identify domains and specific amino acid residues involved in RNA recognition and binding (Webster et al, 1992; Jozwik and Miller, 1992)
Summary
(Received for publication, July 14, 1994, and in revised form, December 18, 1994). Shawn M. 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. More recent studies have focused on structure-function relationships in regA protein in an effort to identify domains and specific amino acid residues involved in RNA recognition and binding (Webster et al, 1992; Jozwik and Miller, 1992). Amino acid similarities to other RNA-binding proteins have been identified for two regions of regA protein; one in the NHz-terminal region between residues Val-15 to His-37 containing a potential helix-turnhelix structural motif (Jozwik and Miller, 1992) and the other in the COOH-terminal region between residues Leu-97 and Lys-113 (Webster et al, 1992) (see Fig. 1). We have examined the sensitivity of regA protein to partial proteolysis by chymotrypsin and trypsin
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