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The cleavage specificity of RNase III
Abstract:
We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.
Insights
RNase III enzyme targets specific sites on lambda cII mRNA when guided by lambda OOP antisense RNA. Enzyme cleavage depends on RNA structure and nucleotide sequence, revealing key specificity factors.
Area of Science:
- Molecular Biology
- RNA Structure and Function
- Enzymology
Background:
- Bacteriophage lambda gene expression is regulated by antisense RNA.
- RNase III is a double-stranded RNA-specific endonuclease involved in RNA processing.
Purpose of the Study:
- To map the precise cleavage sites of RNase III on lambda cII mRNA.
- To investigate the structural and sequence determinants of RNase III specificity in the presence of lambda OOP antisense RNA.
Main Methods:
- Site-directed mutagenesis of lambda OOP RNA to create internal deletions.
- Analysis of lambda cII mRNA cleavage products by RNase III.
- Comparison of cleavage site sequences with a derived consensus sequence.
Main Results:
- RNase III cleaved lambda cII mRNA within double-stranded regions, 10-14 bases from the end of continuous base pairing.
- Cleavage patterns were consistent across different RNA structures with similar double-stranded regions.
- Identified preferred cleavage sites showed good agreement with a consensus sequence, while non-cleavage sites did not.
Conclusions:
- RNase III cleavage specificity is influenced by both the distance from the end of continuous RNA pairing and specific nucleotide sequences.
- The findings provide insights into the mechanism of RNase III in RNA processing and gene regulation.