Structural analysis of the P10/11-P12 RNA domain of yeast RNase P RNA and its interaction with magnesium.

The P10/11-P12 RNA domain of yeast RNase P contains several highly conserved nucleotides within a conserved secondary structure. This RNA domain is essential for enzyme function in vivo, where it has a demonstrated role in divalent cation utilization. To better understand the function of this domain, its structure and alterations in response to magnesium have been investigated in vitro. A secondary structure model of the P10/11-P12 RNA domain had been previously developed by phylogenetic analysis. Computer modeling and energy minimization were applied to the Saccharomyces cerevisiae P10/11-P12 domain to explore alternatives and additional interactions not predicted by the phylogenetic consensus. The working secondary structure models were challenged with data obtained from 1H NMR and in vitro chemical and enzymatic probing experiments. The solution structure of the isolated domain was found to conform to the phylogenetic prediction within the context of the holoenzyme. Structure probing data also discriminated among additional base contacts predicted by energy minimization. The withdrawal of magnesium does not appear to cause gross refolding or rearrangement of the RNA domain structure. Instead, subtle changes occur in the solution accessibility of specific nucleotide positions. Most of the conserved nucleotides reported to be involved in magnesium utilization in vivo also display magnesium-dependent changes in vitro.

Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions.

Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem II.

An intrinsic 22 kDa polypeptide is found associated with the oxygen-evolving photosystem II (PSII) core complex in all green plants and cyanobacteria so far examined, although it does not appear to be required for oxygen evolution. Amino acid sequence information obtained from the purified 22 kDa protein was used to construct a probe that was employed to isolate a full-length cDNA clone encoding the 274-residue precursor of the 22 kDa protein. Hydropathy plot analysis predicts the existence of four membrane-spanning helices in the mature protein. The two halves of the approximately 200-residue mature protein show high sequence similarity to each other, suggesting that the psbS gene arose from an internal gene duplication. The 22 kDa protein has some sequence similarity to chlorophyll a/b-binding proteins.

Tyrosine radicals in photosystem II and related model compounds. Characterization by isotopic labeling and EPR spectroscopy.

Deuteration at selected positions on the phenol ring and at the beta-methylene carbon for the YD.tyrosine radical in Photosystem II in the cyanobacterium Synechocystis 6803 was achieved by growing the organism under conditions in which it is a functional aromatic amino acid auxotroph (Barry, B. A., and Babcock, G. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7099-7103). A series of model tyrosine radicals, also deuterated at specific sites on the aromatic ring and the methylene group, was generated by UV irradiation of frozen solutions. The EPR spectra of the specifically deuterated in vivo radicals confirm that YD.is a tyrosine; nevertheless its spectra differ from those of the tyrosine models. By comparing the EPR spectra of the specifically deuterated radicals with those of the fully protonated, the hyperfine couplings of the various protons of both YD.and the model compound radicals were determined. For both species, the unpaired electron spin density distribution is essentially identical and follows an odd-alternant pattern with high rho values at the carbons ortho and para to the tyrosine phenol oxygen; the meta positions have low spin densities. The differences in EPR spectral characteristics for the two radicals are rationalized as arising from variations in the conformation of the beta-methylene group with respect to the phenol head group. Considering these EPR results and those reported for other model and naturally occurring tyrosine radicals, we conclude that this situation is general; there is little deviation in this class of compounds from the odd-alternant spin density distribution; variations in EPR lineshapes arise primarily from variations in beta-methylene orientation. The conformation of the -CH2- group in biologically active tyrosine radicals deviates from that observed in the models and may be functionally significant. Because the EPR spectrum of YZ., the second redox active tyrosine radical in Photosystem II, is identical to that of YD., we conclude that the two radicals are in similar protein environments, a conclusion that is supported by the protein sequences in the vicinity of the two radicals.