Higher order structure of chloroplastic 5S ribosomal RNA from spinach

Abstract

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.