5-5 RNA from wheat germ, homogeneous by gel filtration and polyacrylamide gel electrophoresis, yields three peaks by hydroxyapatite chromatography. Experiments are presented which firmly establish that the three peaks are constituted by 5-5 RNA molecules ending a t their 5' terminus with mono-, di-, or triphosphate groups. The isolation of 5-S RNA by direct extraction of RNA from wheat embryos homogenized in different buffers and the inability of Escherichia coli alkaline phosphatase to dephosphorylate unmelted 5-S RNA molecules suggests that the three species are not an artifact of extraction.The peculiar ability of the hydroxyapatite chromatography t o fractionate 5-S RNA molecules on the basis of the extent of their 5'-terminal phosphorylation was used to investigate the pattern of 5'-phosphorylation of 5-S RNA from various organisms. The most striking feature observed is that 5-5 RNAs from prokaryotic organisms contain only 5'-monophosphate molecules. I n contrast, the presence of 5' mono-, di-and triphosphate molecules was observed in all the eukaryotic organisms investigated. The significance of this finding in relation to the biosynthesis of 5-5 RNA is discussed.It has been previously shown that ribosomes from all organisms contain a low-molecular-weight RNA of sedimentation constant about 5-5. This RNA is a constituent of the larger ribosomal subunit and its function is a t present unknown [l-91. Recently Nomura and Erdmann [lo] succeeded in reconstituting 50-S ribosomal subunits from Bacillus stearothermophilus from their dissociated molecular components and, using these reconstituted particles lacking 5-5 RNA, they found that 5-5 RNA is essential for the overall activity of 50-S subunits in polypeptide synthesis [ll].The Definition. A,,,~,,,, unit, the quantity of material oontained in 1 ml of a solution which has an absorbance of 1 at 260(271) nm, when measured in a 1-cm pathlength cell.I n a previous paper from this laboratory on wheat germ 5-S RNA we reported that hydroxyapatite chromatography was able to separate pure 5-S preparations in three peaks [17]. On the basis of the behaviour of nucleoside 5'-mOnO-, di-, and triphosphates on hydroxyapatite [18] and of incubation of 5-S RNA with E . coli alkaline phosphatase, we suggested that they differ in the extent of 5'-terminal phosphorylation. The purpose of the present work was to demonstrate the occurrence of pGp, ppGp and pppGp a t the 5'-terminal of wheat germ 5-S RNA and that the three peaks obtained by hydroxyapatite chromatography correspond respectively to the 5'-mono-, di-, and triphosphorylated molecules. Also, using this peculiar ability of the hydroxyapatite chromatography, we have investigated the extent of 5'-terminal phosphorylation of 5-5 RNA from some prokaryotic and eukaryotic organisms. EXPERIMENTAL PROCEDURE MATERIALSE . coli cells (strain ATCC 9637 and FH,,M, an amber mutant lacking alkaline phosphatase) were grown a t 37 "C in a liquid medium containing 0.90/, bacto nutrient broth (Difco) with constant shaking. Pseudomonas fluor...
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