In Vitro Selection of RNA Specifically Cleaved by Bacteriophage T4 RegB Endonuclease

T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional. Not all -GGAG- sites are processed equally by RegB; those found at the Shine−Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sit...

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Bibliographic Details
Published in:Biochemistry (Easton) 1996-02, Vol.35 (7), p.2349-2356
Main Authors: Jayasena, Vineetha K, Brown, David, Shtatland, Timur, Gold, Larry
Format: Article
Language:English
Subjects:
Bacteriophage T4 - metabolism
Base Sequence
DNA Primers
Endoribonucleases - metabolism
Escherichia coli
Hydrolysis
Molecular Sequence Data
Nucleic Acid Conformation
phage T4
RNA - chemistry
RNA - metabolism
RNA Processing, Post-Transcriptional
Substrate Specificity
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Summary:T4 RegB endonuclease specifically cleaves at -GGAG- sites in several early T4 messages, rendering them nonfunctional. Not all -GGAG- sites are processed equally by RegB; those found at the Shine−Dalgarno sequences and in intercistronic regions are processed with higher efficiency than the -GGAG- sites located in coding regions. The low activity of RegB observed in vitro is enhanced by 1−2 orders of magnitude by the Escherichia coli ribosomal protein S1. We have used SELEX (systematic evolution of ligands by exponential enrichment) on a combinatorial RNA library to obtain molecules that are specifically cleaved by T4 RegB endonuclease in the presence of S1. The sequences obtained contain the required -GGAG- tetranucleotide and were unusually enriched in adenosine and cytosine nucleotides. No consensus structure or sequence motif other than -GGAG- was conserved among the selected molecules. The majority of the RNAs are entirely dependent on S1 for RegB-catalyzed cleavage; however, a few RNAs are found to be S1 independent but are cleaved by RegB with much lower rates.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi951879b