To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E). NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates. The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency. Moreover, the loss of single-strandedness was accompanied by a decrease in both the K(m) and k(cat) values.
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Processing of m1 RNA at the 3' end protects its primary transcript from degradation.
J Biol Chem
October 2005
Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5' leader sequence of precursor tRNAs. It is generated by the removal of 36 nucleotides from the 3' end of the primary rnpB transcript (pM1 RNA), but the biological significance of this reaction in bacterial metabolism remains obscure. In this study, we constructed and analyzed bacterial strains carrying mutations in the rne-dependent site of their rnpB genes, showing that the 3' processing of M1 RNA is essential for cell viability.
View Article and Find Full Text PDFKinetic analysis of precursor M1 RNA molecules for exploring substrate specificity of the N-terminal catalytic half of RNase E.
J Biochem
November 2004
Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E). NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates. The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency.
View Article and Find Full Text PDFRole of the sequence of the rne-dependent site in 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P.
FEBS Lett
September 2001
Department of Chemistry and Center for Molecular Design and Synthesis, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea.
The 3' processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps. The precursor of M1 RNA has an rne-dependent site downstream of the processing site, whose sequence variation affects the processing efficiency. In this study, we showed that the sequence itself of the rne-dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites.
View Article and Find Full Text PDFMutational analysis of RNA structures and sequences postulated to affect 3' processing of M1 RNA, the RNA component of Escherichia coli RNase P.
J Biol Chem
August 1996
Department of Chemistry, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea.
When the rnpB gene encoding M1 RNA, the RNA component of Escherichia coli RNase P, is transcribed, the primary M1 RNA transcript (pM1 RNA) is produced and subsequently processed at the 3' end to generate the mature M1 RNA. To study features of pM1 RNA thought to be involved in RNA processing, systematic mutations were introduced in sequence elements and secondary structures surrounding the processing site using p23 RNA, a truncated pM1 RNA transcribed from the internally deleted rnpB gene, as a model substrate and the processing of its mutant derivatives was analyzed in vivo and in vitro. Neither the alteration of two bases forming the processing site nor the disruption of secondary structures surrounding the site significantly affected the processing efficiency although the secondary structures were required for maintaining RNA stability.
View Article and Find Full Text PDFRibonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.
Genes Dev
December 1994
Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università degli Studi di Napoli, Italy.
The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts.
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