The influence of ribosomal proteins on the action of ribosome-inactivating proteins

• Main Author: Romero-Zepeda, Hilda
• Published: University of Warwick 1999
• Subjects: 572.8; QR Microbiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342518

Ribosome-inactivating proteins (RIPs) are produced by many plants and inhibit ribosome function through an N-glycosidase activity that removes a single adenine residue from a universally-conserved stem-loop structure close to the 3' end of the large subunit rRNA. This site specific action is also retained on 'naked' rRNA, but usually with a much lower catalytic efficiency. Although all known RIPs are active on mammalian ribosomes, their activity on ribosomes from other sources varies considerably. In the work reported, the action of two RIPs with different substrate specificities has been studied on ribosomes and sub-ribosomal derivatives from Escherichia coli. The RIPs are pokeweed antiviral protein (PAP), a single chain RIP from the leaves of Phytolacca americana and the catalytically active A-chain (RTA) from the heterodimeric toxic lectin ricin from the endosperm of the castor oil bean. The former RIP is active on native E. coli ribosomes, whereas the latter is inactive but both are active on naked rRNA. Hence, it is postulated that the ribosomal proteins in the native ribosome either allow, in the case of PAP, or prevent, in the case of RTA, action on the target site in the rRNA. The aim of the work is to use ribosome dissociation and reconstitution techniques to study the relationship between ribosomal proteins and the activity of PAP and RTA. The initial part of the work concerned establishing conditions under which both PAP and RTA show high levels of discrimination in activity between native ribosomes and naked rRNA substrates. A buffer that contained Ca2+ in place of the more usual Mg2+ was shown to produce such discrimination. However, in the case of RTA action, the relatively mild treatments resulting in ribosome dissociation were sufficient to allow action. Various subparticles were prepared from purified 50S subunits through the successive removal of ribosomal proteins by increasing ionic strength. As more proteins were split off, the activity of PAP decreased, whereas the activity of RTA remained nearly constant. This is consistent with the hypothesis that ribosomal proteins modulate the activity of the two RIPs differently. In an attemot to use a small defined RNA substrate with which to studv the [influence?] of ribosomal proteins on RIPs' activity the region encoding domain VI of 23S rRNA (containing the RIP target site) was sub-cloned from a cloned rmB operon using PCR and used as a template for the synthesis of transcripts in vitro. These transcripts were susceptible to depurination by PAP, but for unknown reasons were refractory to RTA. Using gel retardation analysis, it was shown that total 50S ribosomal proteins (TP50) bound to domain VI transcripts in an RNA sequence specific manner, and that the reconstituted complex was relatively stable. However, the activity of PAP on this reconstituted RNP was equivalent to that on the transcript alone, and RTA was inactive on both. These results are discussed in relation to the influences of Mg2+ and Ca2+ ions and to the possible role of ribosomal proteins L3 and L6.