Abstract
The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.
Highlights
The bacteriophage T4 RegA protein is a unique translational repressor in that it is able to regulate the expression of 15–30 T4 genes, including its own
As noted by Kang and co-workers (6), two strands of the four-stranded -sheet in domain I of RegA protein exhibit sequence similarities to RNP-1 and RNP-2, suggesting that this region may participate in RNA binding
RNA Binding Domain of RegA Protein photocross-linking of RegA protein to nucleic acid was found to be Phe[106] (8), which lies within domain II
Summary
The bacteriophage T4 RegA protein is a unique translational repressor in that it is able to regulate the expression of 15–30 T4 genes, including its own. Photocross-linking (8), partial proteolysis (9), and truncation studies (9) have been performed on RegA protein, a definitive localization of the RNA binding site on RegA has not been achieved. As noted by Kang and co-workers (6), two strands of the four-stranded -sheet in domain I of RegA protein exhibit sequence similarities to RNP-1 and RNP-2 (including aromatic residues on 5), suggesting that this region may participate in RNA binding. RegA protein has two pairs of basic residues (Lys[7] and Lys[8]; Lys[41] and Lys42) in loops at the base of the -sheet (see Fig. 1), which could be envisioned to function in binding RNA. Partial proteolysis of RegA protein, which leads to cleavage at three sites in the C-terminal domain, is reduced by RNA binding (9)
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