Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

doi:10.1016/0022-2836(74)90526-9

Journal of Molecular Biology

Volume 89, Issue 2, 25 October 1974, Pages 379-382, IN5-IN7, 383-395

https://doi.org/10.1016/0022-2836(74)90526-9 Get rights and content

Abstract

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.

Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.

Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.

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We have studied three sites of 3′ end formation on the Xenopus laevis ribosomal genes, designated T1, T2, and T3. Site T1 colncides with the unique HindIII site at the 3′ end of the 28S sequence and is shown to be a site of rapid RNA processing. Transcription continues another 235 bp past T1 to site T2, which marks a boundary between relatively stable and highly unstable transcripts. However, T2 does not cause polymerase release. In nuclear run-off experiments transcription is detected across the entire spacer until site T3, 215 bp upstream of the gene promoter. T3 shares some sequence homology with T2 but appears to cause polymerase release and is presently the best candidate for a true terminator of transcription on these genes.
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The processing of the high molecular weight primary transcript and intermediates of rRNA have been analyzed after labelling in vivo the oocytes of the blowfly Calliphora erythrocephala which develop in a strictly synchronous manner. The primary 35S transcript ( $2.8 × 10^{6}$ dalton) can be identified after less than 5 min of labelling and is subsequently cleaved, perhaps via a slightly smaller and very short lived 34S intermediate, with a half life time of less than 4 min, to yield the mature 19S rRNA ( $0.7 × 10^{6}$ dalton) and a 29S ( $1.8 × 10^{6}$ dalton) intermediate precursor of the 26S rRNA. The production of the final 26S rRNA, (26S_m), with a molecular weight of $1.44 × 10^{6}$ is mediated by a precursor of $1.49 × 10^{6}$ dalton (27S) with a half life time of some 28 min. Mature 26S rRNA differs from this molecule by a further size reduction of $5 × 10^{4}$ dalton and by an intramolecular cut which generates two hydrogen-bonded submolecules of 26S_m rRNA with estimated molecular weights of $0.6 × 10^{6}$ and $0.78 × 10^{6}$ . They can be separated from one another and from 19S rRNA by polyacrylamide gel electrophoreses after denaturation of the 26S_m rRNA. Besides the “internal break” two further cleavages occur to produce the small 5.8S and 2S submolecules of 26S_m rRNA. These moieties can also be released by thermal denaturation or treatment of 26S_m rRNA with hydrogen-bond interfering agencies. The order of appearance of the primary transcript and intermediates after pulse labelling and the shift of labelling towards fractions of lower molecular weights after application of actinomycin D suggests a scheme for the in vivo processing during the maturation of ribosomal RNA in C. erythrocephala.

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Journal of Molecular Biology

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Abstract

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