Purification of the nuclease responsible for the cleavage in mRNA leader

<p><b>Copyright information:</b></p><p>Taken from "Ribonucleases J1 and J2: two novel endoribonucleases in with functional homology to RNase E"</p><p>Nucleic Acids Research 2005;33(7):2141-2152.</p><p>Published online 14 Apr 2005</p><p>PMCID:PMC1079966.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The leader was transcribed using plasmid pHMS17 linearized by ClaI as template and RNA polymerase (see Materials and Methods). The transcripts, one prematurely terminated at the leader terminator and a read-through transcript, were incubated in 50 μl post-transcriptional assays without the addition of proteins, with 15 μg of a ribosomal high salt wash (HSW) or with the most active enriched protein fraction eluting from a Superdex 200 (S 200) column. Scissors indicate the processed transcript. () Mapping of the processing site shown in (A) in the specifier domain. Primer extension was performed with P-labelled oligonucleotide HP238 on mRNA synthesized in the presence of HSW. Arrows indicate the processing sites within the specifier domain, referred to as cleavage 1 (). A sequencing ladder was run in parallel using the same primer. () Purification of the endoribonucleolytic activity responsible for cleavage in the specifier domain. The most active fractions from the heparin (Hep) and S 200 columns were separated on a 15% SDS–polyacrylamide gel and silver stained. Molecular weight marker proteins (M) were run in the first lane. () Cleavage of the leader transcript by purified YkqC and YmfA proteins. mRNA was prepared as described in (A) and incubated in 50 μl post-transcriptional assays without proteins, with 15 μg of a HSW or with 200 ng of the purified proteins, YmfA, YkqC and a mixture of YmfA and YkqC. The processed transcripts are indicated by scissors and a number (1: cleavage in the AG-box; 2: cleavage near the T-box, see and ). () Cleavage of a leader transcript generated with T7 RNA polymerase from a PCR fragment (see Materials and Methods) by purified YkqC and YmfA proteins. The fully synthesized mRNA was incubated in 10 μl post-transcriptional assays without any proteins or with 200 ng of the purified proteins, YmfA and YkqC. The processed transcripts are indicated by scissors