A physical assay for and kinetic analysis of the interactions between M1 RNA and tRNA precursor substrates

Cecilia Guerrier-Takada, Sidney Altman

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

A gel-shift assay was devised to detect stable enzyme-substrate (E-S) complexes between M1 RNA, the catalytic subunit of RNase P from Escherichia coli, and its tRNA precursor substrates. The use of deletion derivatives of Ml RNA in the gel-shift assay has allowed us to identify regions of the enzyme that are involved in the binding of the substrate or that are necessary for catalytic activity. Fragments of substrates that contain the 3′ CCA sequence bind preferentially to regions in the 5′ half of Ml RNA, while 5′ leader sequences interact primarily with regions in the 3′ half of M1 RNA. The 5′ leader sequence present in the precursor to tRN ATyrsu3 from E. coli plays an important role in the formation of stable E-S complexes with M1 RNA. The CCA sequence at the 3′ end of precursor tRNA substrates is involved in the product-release step of the reaction that is catalyzed by M1 RNA. Direct measurements of the concentrations of all the components in the reaction catalyzed by M1 RNA facilitated a new approach to the kinetic analysis of the action of the enzyme.

Original languageEnglish (US)
Pages (from-to)7152-7161
Number of pages10
JournalBiochemistry
Volume32
Issue number28
StatePublished - 1993
Externally publishedYes

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RNA Precursors
Assays
RNA
Kinetics
Substrates
Escherichia coli
Enzymes
Gels
Ribonuclease P
Catalytic RNA
Catalyst activity
Catalytic Domain
Derivatives

ASJC Scopus subject areas

  • Biochemistry

Cite this

A physical assay for and kinetic analysis of the interactions between M1 RNA and tRNA precursor substrates. / Guerrier-Takada, Cecilia; Altman, Sidney.

In: Biochemistry, Vol. 32, No. 28, 1993, p. 7152-7161.

Research output: Contribution to journalArticle

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