Volume 16 Number 4 1988                                                                Nucleic Acids Research

Accurate in vitro cleavage by RNase III of phosphorothioate-substituted RNA processing signals in
bacteriophage T7 early mRNA

Allen W. Nicholson, Kenneth R. Niebling*, Paul L. McOsker*, and Hugh D. Robertson*

Department of Biological Sciences, Wayne State University, Detroit, MI 48202 and the *Rockefeller University, New York, NY 10021, USA

ABSTRACT

     To test the ability of an RNA processing enzyme to cleave chemically-
modified RNA substrates, RNA transcripts containing RNase III cleavage sites
were enzymatically synthesized in vitro to contain specific phosphorothioate
diester internucleotide linkages.  One transcript (R1.1 RNA) was generated
using phage T7 RNA polymerase and a cloned segment of phage T7 DNA
containing the R1.1 RNase III processing site.  The second transcript was
the phage T7 polycistronic early mRNA precursor, which was synthezsized
using E. coli RNA polymerase and T7 genomic DNA.  The RNA transcripts
contained phosphorothioate diester groups at positions including the
scissile bonds.  The modified RNAs were stable to incubation in Mg2+ 
containing buffers, and were specifically cleaved by RNase III.  RNA 
oligonucleotide sequence  analysis showed that the modified R1.1 RNA
processing site was the  same as the cononical site and contained a 
phosphorothioate bond.  Furthermore,  RNase III cleaved the phosphoro-
thioate internucleotide bond with 5' polarity.  RNase III  cleavage of
phosphorothioate substituted T7 polycistronic early mRNA precursor
produced the same gel electrophoretic pattern as that obtained with the
control transcript.  Thus,  RNase III cleavage specificity is not altered by
phosphorothioate internucleotide linkages.