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.

No abstract

Fluorescence of quinacrine in the presence of different polynucleotides was studied to attempt to identify the specific nucleotides responsible for the fluorescence of stained chromosome preparations. A marked enhancement of fluorescence was seen in the presence of bihelical polynucleotides, such as poly(dA-dT), poly(dA) poly(dT), and poly(rA) poly(rU), but not in the presence of single-stranded polynucleotides, such as poly(dA), poly(dT), poly(rA), or poly(rU) alone. The higher was the GC content of natural DNAs, the more they quenched. Quenching was also seen with poly(dG) or poly(rG) alone, but not with poly(dC) or poly(rC) alone.Native and denatured DNA were both effective in quenching fluorescence. Thus, a bihelical conformation is not required for fluorescence quenching. Nearly all of these properties are shared with proflavine. In contrast, acridine orange, which stains most areas of chromosome preparations, shows enhanced fluorescence in the presence of all members of a series of natural DNAs. These data suggest that base-pairs composed of AT (rather than GC) residues are responsible for the observed fluorescence of specific chromosome regions after treatment with quinacrine, and support the proposal of Ellison and Barr (Chromosoma, in press) that the highly localized quinacrine fluorescence in their cytological preparations reflects the presence of DNA that has a high (A + T)/(G + C) ratio.Quinacrine mustard was shown by Caspersson and his colleagues (1) to stain certain regions of chromosomes with a very brilliant intensity. It was proposed that the mustard group might react with high specificity with the reactive N-7 position of guanine, thereby providing an affinity label for GC base-pairs in DNA. It was subsequently observed by Vosa (2) and others, however, that quinacrine itself possessed the same specificity as its mustard derivative. This finding suggested that the mustard function was not critical for the specificity of the staining reaction.The base specificity of this reaction was not further elucidated until Ellison and Barr (3) compounds that might exhibit similar staining properties with fluorescent dyes, and from which the base specificity of the quinacrine staining reaction might be extrapolated. MATERIALS AND METHODSRibopolynucleotides, Clostridium perfringens DNA, Escherichia coli DNA, and Micrococcus luteus DNA were purchased from the Sigma Chemical Co. Poly(dA), poly(dT), poly(dG), and poly(dC) were generously donated by R. D. Wells (Biochemistry Dept., Univ. of Wis.). Poly (dA-dT) was synthesized (4). Agrobacter tumefaciens DNA was prepared from A. tumefaciens (strain B6) by the method of Schilperoort (5). Chicken DNA was prepared from erythrocyte nuclei by treatment with Pronase and sodium dodecyl sulfate, by the same method used to purify A. tumefaciens DNA. Quinacrine * HC1 was obtained from the Sigma Chemical Co., and acridine orange was a product of the Chroma-Gesellschaft, Schmid and Co.All reactions were performed in 0.1 M Na phosphate buffer (pH 6.8). Fluorescence...

Local determinants of 3,0-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 5 1 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3,0-helices and multipleturn conformations, had strong amino acid sequence patterns not evident among a-helices. Aspartate occurred over twice as frequently in the N-cap position of 310-helices as in the N-cap position of a-helices. Unlike a-helices, 310-helices had few C-termini ending in a left-handed a conformation; most 310 C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3,0-and a-helices were also apparent, producing amphipathic 310-helices. Local interactions that stabilize 3,0-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of a-helices have been postulated to proceed through reverse-turn and 310-helix intermediates, sequence differences between 310-and ahelices can also lend insight into factors influencing a-helix initiation and propagation. Keywords: a-helix; amino acid determinants; 310-helicesThe conformation of a segment of protein backbone is determined both by local interactions, i.e., interactions within the segment and with residues neighboring in sequence, and by interactions with the segment's tertiary environment. We have undertaken a series of studies of three-dimensional protein crystal structures, involving the systematic classification of local protein conformations and the analysis of correlations between amino acid sequence and local conformation classes (Karpen, 1991). Strong correlations between amino acid sequence and local conformation can point to side-chain interactions that stabilize or destabilize a particular conformation relative to other conformations. In this paper, we examine 310-helices and how their amino acid sequences differ from a-helices. The 310-helical structures are relatively common in proteins; 4% of all residues in our select data base of 51 high-resolution proteins are involved in 310-helices. These short helices usually occur on the surface of a pro-