No abstract

The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.

The affinity and stoichiometry of DNA binding by Escherichia coli tip repressor were studied by electrophoresis in nondenaturing gels. The ability of trp repressor to retard the electrophoretic mobility of an operator DNA fragment depends on the pH of the gel system. Above the pI of the protein, little retardation of DNA is observed, although complex formation can be detected by other assays. As the pH of the gel is lowered, retardation is enhanced. The apparent dissociation constant for the interaction between hp repressor and trpEDCBA operator fragments is 0.5 nM under the conditions used here. Nonspecific binding occurs with only about 200-fold weaker affinity. The stoichiometries of specific and nonspecific complexes were determined directly by using tip repressor labeled in vivo. High-affinity operator binding requires a single dimer of tip repressor. DNase I-protection analysis ("footprinting") was used to confirm the dissociation constants and to locate the binding site.Escherichia coli trp repressor (TrpR) regulates transcription initiation at three operons involved in tryptophan biosynthesis: trpEDCBA (1), aroH (2), and trpR (3). Operator sites have been identified near the transcription initiation points by homology of the three DNA sequences. Repressor is a dimer of identical 12.5-kDa subunits (4). The crystal structure shows that the protein is all a-helical (5) and each subunit contains a helix-turn-helix supersecondary structure (6), which mutational analysis (7) suggests is the site of DNA binding. The subunits are in intimate contact over a large area and are nearly interwound (5). Unlike many repressors, TrpR has an acidic isoelectric point (4), has fewer polar and positively charged amino acids in the DNA-binding domain (5), and requires the binding of two molecules of Ltryptophan per dimer for operator-specific DNA binding (8). Each tryptophan binding site comprises residues from both subunits (5). Binding of L-tryptophan induces a local conformational change that repositions the two DNA-binding domains of the dimer so that they exactly align with two successive major grooves of the DNA (9). DNA binding by TrpR has been studied primarily by an indirect assay (3, 4) in which bound protein prevents the restriction enzyme Rsa I from cleaving a site in the operator. To begin to understand the biochemical basis for DNA binding to TrpR, I have used a direct quantitative assay to determine the binding constants and stoichiometry of the interaction.Electrophoresis in nondenaturing gels has been used for nearly two decades to study ribonucleoproteins (10). The current popularity of the technique dates from 1981, when Garner and Revzin (11) and Fried and Crothers (12) applied it to detect protein-DNA interactions. The method is based on the observation that the electrophoretic mobility of a polynucleotide is reduced when a protein is bound to it.Binding proteins can be detected in and purified from crude extracts by their retardation activity. The potential to separate complexes that differ only...

The crystal structure of trp repressor tandemly bound in a 2:1 complex to a 16-base-pair palindromic DNA containing a central trp operator half-site has been determined and refined to 2.4 A resolution. Despite dramatically different DNA sequence contexts and crystallization conditions, the protein/DNA interface is essentially identical to that seen in the original trp repressor/operator complex structure. Water-mediated sequence recognition by trp repressor is likely to be related to the unusual end-on approach of the recognition helix (E), which allows sharing of the major groove by tandem dimers. The tandem complex model accounts for the mutational sensitivity of all trp operator base pairs. The structure also provides the first detailed view of the tandem interaction, revealing a key role for the amino-terminal arms.