ABSTRACTEscherichia colicontains multiple 3′ to 5′ RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3′ end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.