Characterization in vitro of the defect in a temperature-sensitive mutant of the protein subunit of RNase P from Escherichia coli

M. F. Baer, D. Wesolowski, S. Altman

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

We have studied the assembly of Escherichia coli RNAse P from its catalytic RNA subunit (M1 RNA) and its protein subunit C5 protein). A mutant form of the protein subunit, C5(A49), has been purified to apparent homogeneity from a strain of E. coli carrying a thermosensitive mutation in the rnpA gene. The heat stability. However, when the catalytic efficiencies of the holoenzymes were compared, we found that the holoenzyme containing the mutant protein had a lower efficiency of cleavage than the wild-type holoenzyme at 33, 37, and 44°C. We then explored the interaction of M1 RNA and C5 protein during the assembly of the holoenzyme. The yield of active holoenzyme obtained by reconstitution with wild-type M1 RNA and C5(A49) protein in vitro can be considerably enhanced by the addition of excess M1 RNA, just as it can be in vivo. We concluded that the Arg-46 → His-46 mutation in the C5(A49) protein affects the ability of the protein to participate with M1 RNA in the normal assembly process of RNase P.

Original languageEnglish (US)
Pages (from-to)6862-6866
Number of pages5
JournalJournal of bacteriology
Volume171
Issue number12
DOIs
StatePublished - Jan 1 1989
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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