In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs

Michele Yip-Schneider, M. J. Holland

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.

Original languageEnglish (US)
Pages (from-to)4045-4051
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number7
StatePublished - 1989
Externally publishedYes

Fingerprint

Ribosomal RNA
Yeast
Yeasts
RNA
Cell Extracts
Transcription
Post Transcriptional RNA Processing
Processing
RNA Polymerase I
Micrococcal Nuclease
Ribonucleotides
Endopeptidase K
RNA Precursors
Saccharomyces cerevisiae
Nucleotides
Molecules
Genes
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

In vitro RNA processing generates mature 3' termini of yeast 35 and 25 S ribosomal RNAs. / Yip-Schneider, Michele; Holland, M. J.

In: Journal of Biological Chemistry, Vol. 264, No. 7, 1989, p. 4045-4051.

Research output: Contribution to journalArticle

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N2 - 35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.

AB - 35 S rRNA is the major intracellular precursor to 18, 5.8, and 25 S rRNAs in Saccharomyces cerevisiae. In this report, we show that the 3' termini of 35 S rRNA as well as 25 S rRNA are generated by post-transcriptional RNA processing rather than transcription termination. Using a partially purified yeast whole cell extract, efficient site-specific cleavage of a synthetic rRNA precursor was demonstrated in vitro. The 3' termini of the processed precursor were established by S1 nuclease protection analysis. RNA molecules containing the mature 3' termini of 35 and 25 S rRNA as well as molecules with a 3' terminus located 12 nucleotides beyond the 3' terminus of 25 S rRNA were the major products of the in vitro processing reaction. Processing activity required Mg2+ but was independent of ribonucleotides. Pretreatment of the yeast whole cell extract with proteinase K abolished processing activity, whereas micrococcal nuclease pretreatment of the extract had no effect on processing activity. These results show that RNA polymerase I-dependent transcription of yeast ribosomal cistrons continues beyond sequences that encode the 3' terminus of 35 S rRNA into the spacer region that separates 35 S rRNA transcription units.

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