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- 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.
Jayasena
V K
VK
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347, USA.
Brown
D
D
Shtatland
T
T
Gold
L
L
D017122:Bacteriophage T4 / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D013379:Substrate Specificity
D017931:DNA Primers; D012313:RNA; D004722:Endoribonucleases