Definition of the molecular forces which contribute to the specificity and stability in protein-nuclei acid assemblies is essential to obtaining a molecular-level understanding of gene expression. Bacteriophage T4 gene 32 protein (g32P) is an excellent experimental vehicle for this objective. The classical binding by g32P is a highly cooperative """"""""nonspecific"""""""" single- stranded (ss) DNA binding activity which functions in T4 DNA replication, recombination and repair. In addition, g32P displays a """"""""sequence""""""""-specific recognition in autogenous translational regulation of g32P biosynthesis. G32P also contains a cysteine-sulfur rich """"""""zinc-finger"""""""" Zn(II) domain which can potentially modulate these binding activities. The structure and functional importance of the Zn(II) coordination domain in g32P is in need of further definition. A quantitative determination of how depletion of the intrinsic metal ion from g32P perturbs the thermodynamics of protein binding to both idealized ssDNA and RNA homopolynucleotide substrates and select synthetic translational operator RNA fragments will be undertaken. Reconstitution of T4 replication in vitro with metal-free g32P will define any functional defect in the replicative accessory activity of g32P. To substantiate the current model for the Zn(II) coordination domain in g32P, conventional and proton-detected heteronuclear (113Cd and 15N) NMR spectroscopy of isotopically-enriched wild-type and mutant g32Ps is in progress to complete the identification of the Zn(II) liganding atoms. Finally, the energetics of cooperative binding by g32P remain poorly defined. This will be approached by the introduction of a large set of single amino acid substitutions at residues 2-8 in order to identify the functionally important side chains in the cooperative binding mode. A companion biological screening assay based on translational autogeny in vivo is described. This assay will quickly identify g32P N-terminal mutants functionally deficient but not abolished in cooperative binding for which quantitative nucleic acid binding parameters will be obtained and compared to the wild-type protein in vitro. The systematic approach outlined here for g32P should be directly applicable to the future study of structurally related zinc-containing nonspecific single-stranded RNA binding proteins which encapsidate viral genomic RNA in exogenous human retroviruses, including the etiologic agents of adult T-cell leukemia and AIDS.
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