rRNA protection: MBP-Nmd3 interaction with helices H38, H65, H69, and H95 of 25S. (A) 60S subunits were incubated with no protein, MBP-Nmd3, or GST-Nmd3 and treated with RNaseV1. The rRNA was extracted, and primer extension reactions were performed to identify regions of altered sensitivity to RNaseV1. Sequencing reaction lanes are marked by the dideoxynucleotide present in the mixture. Primers used were H38, AJO1061; H65, AJO501; H69, AJO1060; and H95, AJO1135. Numbers indicate positions (E. coli numbering) of nucleotides showing major alteration in sensitivity to RNaseV1. Unp, unprotected (no Nmd3); MBP, MBP-Nmd3; GST, GST-Nmd3. (B) An interface view of the 50S subunit of the 70S E. coli crystal structure (PDB ID 2AW4) showing the position of the helices concerned. Nucleotides marked in green are protected from RNaseV1 by Nmd3; nucleotides in purple show enhancement of cleavage upon interaction with Nmd3; the nucleotide in red shows protection from RNaseV1 cleavage by GST; and the nucleotide in yellow is protected from RNaseV1 cleavage by MBP.