Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model

Abstract

Ricin produced by the castor bean plant and Shiga toxins produced by pathogenic Escherichia coli (STEC) and Shigella dysenteriae are type II ribosome inactivating proteins (RIPs), containing an enzymatically active A subunit that inhibits protein synthesis by removing an adenine from the a-sarcin/ricin loop (SRL) of the 28S rRNA. There are currently no known antidotes to Shiga toxin or ricin, and the ability to screen large chemical libraries for inhibitors has been hindered by lack of quantitative assays for catalytic activity that can be adapted to a high throughput format. Here, we describe the development of a robust and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that can directly measure the toxins' catalytic activity on ribosomes and can be used to examine the kinetics of depurination in vivo. The qRT-PCR assay exhibited a much wider dynamic range than the previously used primer extension assay (500-fold vs. 16-fold) and increased sensitivity (60 pM vs. 0.57 nM). Using this assay, a 400-fold increase in ribosome depurination was observed in yeast expressing ricin A chain (RTA) relative to uninduced cells. Pteroic acid, a known inhibitor of enzymatic activity, inhibited ribosome depurination by RTA and Shiga toxin 2 with an IC 50 of~100 mM, while inhibitors of ricin transport failed to inhibit catalytic activity. These results demonstrate that the qRT-PCR assay would enable refined kinetic studies with RIPs and could be a powerful screening tool to identify inhibitors of catalytic activity.

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome

“…The 25S reference primers were 5=-AGA CCG TCG CTT GCT ACA AT-3= and 5=-ATG ACG AGG CAT TTG GCT AC-3=, and the depurination primers were 5=-CTA TCG ATC CTT TAG TCC CTC-3= and 5=-CCG AAT GAA CTG TTC CAC A-3=, respectively. The comparative threshold (⌬⌬C T ) method was used to calculate the depurination levels, and data were expressed as a fold change of depurination in Stx-treated RNA over depurination in control nontreated RNA, as described previously (30).…”

“…RNA was extracted with phenol and then phenol-chloroform and precipitated overnight with ethanol. Depurination was determined using quantitative reverse transcriptase PCR (qRT-PCR) (30).…”

“…RNA was purified with phenol and then phenol-chloroform, and the depurination of rRNA was quantified using a qRT-PCR assay. The ⌬⌬C T method was used to calculate the depurination level, and data were expressed as the fold change of depurination in Stx-treated RNA over depurination in nontreated RNA, as described previously (30).…”

“…Total RNA was extracted using the RNeasy minikit (Qiagen, Valencia, CA) with on-column DNase treatment. A qRT-PCR assay was used to quantify depurination (30).…”

“…One nanomolar WT Stx1A1 and Stx2A1 and 3-fold dilutions (9 nM, 3 nM, and 1 nM) of mutant proteins (R170A, R176A, and R172A/R176A) were used in the treatment of 7 pmol of rat ribosomes. Depurination was analyzed by qRT-PCR (30).…”