A FLASH of insight into cellular chemistry: genetically encoded labels for protein visualization in vivo.

Genetically encoded fluorescent labels, such as green fluorescent protein, make it possible to visualize a protein's natural distribution and environment in living cells. A new approach to protein labeling in living cells has been devised in which a small, membrane-permeable ligand binds with high affinity and specificity to a short peptide motif that can be incorporated into the protein of interest; the ligand becomes brightly fluorescent after binding to the peptide.

Solution conformation of a five-nucleotide RNA bulge loop from a group I intron.

We present the solution conformation, determined by NMR spectroscopy, of a five-nucleotide RNA bulge loop. The bulge interrupts the stem of a 25-nucleotide RNA hairpin, and its sequence and flanking sequences are those of a conserved bulge from a Group I intron. The secondary structure of the bulge loop in the hairpin context is that predicted by the secondary structure prediction algorithm of Zuker. It differs, however, from the secondary structure deduced from sequence covariation of the bulge in the context of the functionally folded Group I introns and observed in the crystal structure of an independently folding domain of the Group I intron from Tetrahymena thermophila. This difference represents an exception to the heierarchical model of RNA folding in which preformed elements of secondary structure interact to form a tertiary structure. The three-dimensional structure of the bulge loop is characterized by discontinuous base stacking. Adjacent adenines stack with each other and with the flanking double helices. However, the position of the central uracil is not well defined by NOE distance constraints and is a point of discontinuity in the base stacking.

Sequence effects on RNA bulge-induced helix bending and a conserved five-nucleotide bulge from the group I introns.

Bulge loops introduce bends in RNA double helices. Thus, a role for bulge loops in the tertiary folding of RNA is to orient helical elements. The location, size, and sequence of a five-nucleotide bulge are conserved in many of the self-splicing group I introns. We have used gel electrophoretic analysis of helix bending to test the hypothesis that this bulge loop is conserved to control the angle between the flanking helices. Interruption of an RNA duplex by the five-nucleotide bulge of the group I intron from Tetrahymena thermophila results in an electrophoretically retarded species, indicative of bending by the bulge. However, mutation of conserved bases in the bulge has a small effect on the retardation, suggesting that the average induced bend angle is not strongly dependent on the conserved sequence. Electrophoretic analysis of a mixture of bulged duplexes containing all five-nucleotide bulges reveals that most five-nucleotide bulge sequences induce bends that are similar to the bend induced by the conserved bulge. We have calibrated relative electrophoretic mobilities with bends of known magnitude, and characterized the distribution of bulge sequences among bend angles. Though the entire range of bend angles induced by different five-nucleotide bulges is from approximately 45 degrees to 75 degrees, most ( > 85%) five-nucleotide bulge loops induce bends between 65 degrees and 75 degrees. We have identified several of the anomalous five-nucleotide bulge sequences that induce bends of magnitude smaller than 65 degrees. They are generally, though not universally, pyrimidine-rich.

Nonenymatic ligation of double-helical DNA by alternate-strand triple helix formation.

Nonenzymatic ligation of double-stranded DNA has been performed using an alternate-strand binding oligodeoxyribonucleotide template to juxtapose the duplex termini in a triple helical complex. The template associates with the duplex termini by Hoogsteen hydrogen bonding to alternate strands on opposite sides of the ligation site. Intermolecular and intramolecular ligation of linearized plasmid DNA are observed in the reaction, which depends on the template oligodeoxyribonucleotide and a condensing agent, N-cyanoimidazole. Intramolecular ligation products include those in which both strands are covalently closed in a circle. Ligation of the two strands is sequential and occurs at comparable rates for the first and second strands ligating. The covalent linkages formed in the reaction can be cleaved by the restriction endonuclease Stu I, supporting their identification as phosphodiesters.

Photochemically induced binding of Rh(phen)2Cl2+ to DNA.

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.