On the conformation of the anticodon loops of initiator and elongator methionine tRNAs.

The solution conformations of analogues of initiator and elongator tRNA anticodon stem-loops have been compared by NMR. The data indicate that both have conformations closely similar to those reported for crystalline elongator tRNAs. The two loops differ in their dynamics, however: that of the elongator analogue is more flexible than its initiator counterpart. The anticodon stem-loops of initiator tRNAs are more likely to be distinguished from those of elongator tRNAs during initiation on the basis of their distinctive stem sequences, than they are by differences in the conformations of their anticodon loops.

Structural characterization of a ribonuclease III processing signal.

The structure of a ribonuclease III processing signal from bacteriophage T7 was examined by NMR spectroscopy, optical melting, and chemical and enzymatic modification. A 41 nucleotide variant of the T7 R1.1 processing signal has two Watson-Crick base-paired helices separated by an internal loop, consistent with its predicted secondary structure. The internal loop is neither rigidly structured nor completely exposed to solvent, and seems to be helical. The secondary structure of R1.1 RNA is largely insensitive to the monovalent cation concentration, which suggests that the monovalent cation sensitivity of secondary site cleavage by RNase III is not due to a low salt-induced RNA conformational change. However, spectroscopic data show that Mg2+ affects the conformation of the internal loop, suggesting a divalent cation binding site(s) within this region. The Mg(2+)-dependence of RNase III processing of some substrates may reflect not only a requirement for a divalent cation as a catalytic cofactor, but also a requirement for a local RNA conformation which is divalent cation-stabilized.

Evaluation of plasma membrane stability by detergent-induced rupture of osmotically swollen sperm.

A general assay for plasma membrane stability was developed and tested. Osmotically swollen spermatozoa were ruptured with detergents and their volume distribution was monitored with resistance pulse spectroscopy. The extent of cell breakage was determined and expressed as [D]50, the concentration of detergent necessary to lyse 50% of the initially intact cells. Preliminary experiments established the degree to which spermatozoa could be swollen without lysis (no detergent) and the ability of the method to detect known mixtures of intact and membrane disrupted spermatozoa. [D]50 values were determined for caput (immature) and cauda (mature) ram epididymal spermatozoa with four detergents (cetyltrimethylammonium bromide, sodium dodecylsulfate, Zwittergent 3-14, and sodium deoxycholate). [D]50 values for caput spermatozoa were higher than those for cauda spermatozoa (P less than 0.05) for all detergents but cetyltrimethylammonium bromide. These changes are consistent with a qualitative model of membrane structure and stability based on lipid shape and composition and with the compositional changes known to occur during epididymal maturation. Additional studies using rooster spermatozoa established that a typical cryopreservation protocol leaves the surviving spermatozoa with membranes with greater sensitivity to detergent-induced stress. Since osmotic swelling has been microscopically localized to the tail plasma membrane, the changes in membrane stability can be assigned specifically to that region.