RNase inhibitors and RNases

Question  

1.What are the differences among RNase H, RNase A, RNase B and RNase C?

2.In your cDNA kits, RNase H is added in the second strand reaction to produce more nicked RNA as primers for DNA synthesis. In this repect, would RNase out RNase inhibitor influence the function of RNase H, if it was added even before first stand synthesis?

Answer  

1. The main difference between all RNases are where they cleave the RNA (what site they recognize) and whether it is single stranded or double stranded.

RNase H is an endoribonuclease that specifically hydorlyzes the phosphodiester bonds of RNA in RNA:DNA duplexes to generate products with 3' hydroxyl and 5' phosphate ends. It will not degrade single-stranded or double-stranded DNA or RNA.

RNase A is an endoribonuclease that specifically hydrolyzes RNA after C and U residues. Cleavage occurs between the 3'-phosphate group of a pyrimidine ribonucleotide and the 5'-hydroxyl of the adjacent nucleotide. The reaction generates a 2':3' cyclic phosphate which then is hydrolyzed to the corresponding 3'nucleoside phosphates.

RNase B is a glycoprotein which possesses an amino acid composition indistinguishable from that of RNase A and which contains carbohydrate to the extent of 6 residues of mannose and 2 residues of N-acetylglucosamine per molecule. It is consequently considered to be a carbohydrate derivative of RNase A. ( Tarentino, A., Plummer, J., and Maley, F.: Studies on the Oligosaccharide Sequence of Ribonuclease B, J. Biol. Chem., 245, 4150 (1970).)
RNase B has the same specificity as RNase A. (Plummer, T., and Hirs, C.: The Isolation of Ribonuclease B, a Glycoprotein From Bovine Pancreatic Juice, J. Biol. Chem., 238, 1396 (1963).)


2. RNASEOUT?inhibits RNase A, B, and C but does not inhibit RNase 1, RNase T1, S1 Nuclease, RNase H, RNase T2. So adding it in first strand will not cause a problem using RNase H in second strand synthesis.