5‐S RNA: Investigation of the Different Extent of Phosphorylation at 5′ Terminus

Abstract

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...

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

“…The resolution of isolated 5s RNA into three fractions by hydroxyapatite chromatography is in accordance to the findings of Soave et al [59] who first reported a differing number of 5'-terminal phosphates. We confirmed this variable number of 5'-terminal phosphates using alkaline phosphatase, which dephosphorylated the 5'-nucleotides to the mono-or dephosphorylated nucleoside and abolished the chromatographic heterogeneity.…”

“…RNA III from both RNPs exhibited an increased lability at position 43 and a weak susceptibility of position G69 to single-strand-specific RNase. All other S1 nuclease and CSV RNase cuts were in line with the current 5s rRNA secondary structure model and as found in other eukaryotic RNAs [30, 551. A separation of isolated 5s rRNAs into three peaks upon hydroxyapatite chromatography has been previously shown to be due to variations in the number of phosphates remaining on the 5'-nucleotide [59]. This was confirmed here for the isolated 5s RNA from both sarcoplasmic and liver ribosomal RNPs by alkaline phosphatase treatment (Fig.…”

Turnover of the 5′‐end phosphate in yeast tRNA in vivo

“…During the alkaline hydrolysis any nucleoside retains at its 3'(2')-position a phosphate group originating from 5'-position of its neighbouring nucleoside. It is worth mentioning that an independent turnover of the 5'-phosphate end of 5 S rRNA in rabbit reticulocytes was not shown in [14]. As shown in table 1, pGp from the 5'-end of tRNA exhibits higher specific activity than the 3 nucleotides obtained from internal positions of the polynucleotide chains.…”

Triphosphate residues at the 5' ends of rRNA precursor and 5S RNA from Dictyostelium discoideum.

“…Thus, bacterial 5S RNAs generally bear a 5'-terminal pU-(or, rarely, pC-) (35), consistent with the processing of these RNAs from larger precursors. In contrast, the 5'-end groups of several eukaryotic 5S RNAs have been shown to be triphosphate residues, almost always pppG- (26)(27)(28)36), although at least one algal 5S RNA is terminated with pppA- (37). In animals (9)(10)(11)(12), and also in the protozoan Tetrahymena (13), independent transcription of 5S RNA is insured by the complete lack of linkage between 5S DNA and rDNA.…”