Determination of the angle between the anticodon and aminoacyl acceptor stems of yeast phenylalanyl tRNA in solution.

A principal feature of the crystal structures of tRNAs is an L-shaped tertiary conformation in which the aminoacyl acceptor stem and the anticodon stem are approximately perpendicular. However, the anticodon-acceptor interstem angle has not been precisely quantified in solution for any tRNA. Such a determination would represent an important test of the predicted global conformation of tRNAs in solution. To this end, we have constructed a yeast tRNA(Phe) heteroduplex RNA molecule in which the anticodon and acceptor stems of the tRNA have each been extended by approximately 70 base pairs. A comparison of the rotational decay times of the heteroduplex molecule and a linear control yields an interstem angle of 89 +/- 4 degrees in 4 mM magnesium chloride/100 microM spermine hydrochloride, essentially identical to the corresponding angle observed in the crystal under similar buffer and temperature conditions. The current approach is applicable to the study of a wide variety of RNA molecules that possess elements of nonhelical structure.

Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa.

During fertilization in marine invertebrates, fusion between sperm and egg cell membranes occurs at the tip of the sperm acrosomal process. In abalone sperm the acrosomal process is coated with an 18-kDa protein. In situ, this protein has no effect on the egg vitelline envelope, but in vitro it is a potent fusagen of liposomes. Thus, the 18-kDa protein may mediate membrane fusion between the gametes, a step in gamete recognition known to restrict heterospecific fertilization in other species. The cDNA and deduced amino acid sequences of the 18-kDa protein were determined for five species of California abalone. The deduced amino acid sequences exhibit extraordinary divergence; the percent identity varies from 27% to 87%. Analysis of nucleotide substitution shows extremely high frequencies of amino acid-altering substitution compared to silent substitution, demonstrating that positive Darwinian selection promotes the divergence of this protein. However, amino acid replacement is conservative with respect to size and polarity of residue. The data support the developing idea that in free-spawning marine invertebrates, the proteins mediating fertilization may be subjected to intense, and as yet unknown, selective forces. The extraordinary divergence of fertilization proteins may be related to the establishment of barriers to heterospecific fertilization.