Binding of RNA by the alfalfa mosaic virus movement protein is biphasic

Abstract

The movcmcnt protein of ;rlfal~I mosaic virus wus cxprcsscd in IzrlrrrirlrL ruli tend purified by cation cxchangc chromatography. The purifisd proicin bound singlestranded RPJA caopcntivtly in u biphusic manner. At protein sa~untion. RNAlprotcin cornplcxe (dnigttrtcd 'primnry complexa') wcrc dctatcd by a Ilitroccllulor-ntcntion assay within I min of mixing. bath at 4 and 2X. In contrast, rn incubation of 30 min UI 22.C was nacssary to obtain clectrophorcticolly retarded complcxcs 0ubilizcd camplcxn'), containing a large number of protein moloculcs bound stably to wcli molcculc of RNA. Stabilization did not take plvcc ut 4.C. The nlc of formelien of the primary complclra was rrronSly dcpcn&nt on protein conerntntion. nnd thus appeared limited by u bimolecular inlcnclian. fn contmsl. the rate of stabilization was indcpcndcnt of protein canccntrntion. ruacstiny thttt this process rcmrirtcd oTa rcnrranpmcnt of the primary complcxcs without binding of udditionttl protein molccula, In uBrccmcnt with this rul(g#tion. tht ilmount ofcomplcxcd RNA at equilibrium wus the UI~P when rssnycd by nitroccllulorr: rctcntion nnd by clcctrophorctie rctardrtion. The pasribility thpl these pxuliar kinetics could bccauscd by the prcrensc of Twccn 20 in the incubation mcdiu is discussed. BLII(DE3) cells carrying the rccambinunt pET3d vectors wcrc grown overnight at 37'C in 20 ml LB medium contflining I50 p@ml ampicillin. The culture was diluted twofold with LB ntcdium and another I SO,u#ml of ampicillin WBS uddcd to m&c up for rhc loss due IO the ~.laclumasc rclcxcd during overnight growlh, IPTG (0.4 mM) was added to induce expression of thcchimcric gcnf end the cells were grown for ar,othcr 3 h, The bactcriu wcrc harvcstcd by ccntrifuption and resuspendal in S ml bulTcr A (50 mM Tris+lCl, pH 7.550 mM NKI. 5% glycerol. IO mM EDTA. IO mM 2~mcrraprocthanol) containing 0,4 mylmi lynozymc nnd I mM phcnylmcthylrulfonyi iiucr+!! IPMSF), After 20 min of incr:hation at 37*C. they wcrc rubmittcd to 3 or 4 frccz-th;lw cycles. The inclusion bodies and ccl1 dcbrir wcrc pcllclcd by ccntrifugution at 5.0OO x x for 10 min and rctxtractcd with I.5 ml bulTcr A. then with I,5 ml buifcr B (SO mM Trir-HCI. pH 7.5. IO mM EDTA. 10 mM 20mercaprocthrtnol) containing 1% Nonidct P40 and I mM PMSF, then wtlohcd twice with I.5 ml bulTcr C (SO mM sodium acckttc, pH 5.5, IO mM 2-mcrcaptocthanol). The recombinant protein was solub&cd with I.5 ml bulTcr C contuining 6 M urcil. purified by chromatoyrnphy on cnrboxymcthyl Trisncryl nnd rcnaturcd us dca ssrilxd [II, 11 was atorcdat -80°C in 50%glyccrol, Theconcentration

Transient Structural Ordering of the RNA‐Binding Domain of Carnation Mottle Virus p7 Movement Protein Modulates Nucleic Acid Binding

“…Most of the MPs that bind to ssRNA also have ssDNA-binding properties. [22,[34][35][36] Thus, in an attempt to bypass the problems of RNA-protein aggregation and to obtain a more accurate quantitative description of the binding process, [37] we used short ssDNA of different lengths. In this way we avoided the multiple binding of proteins to a single nucleic acid molecule that was probably the cause of the observed complex insolubility.…”

Effect of the alfalfa mosaic virus movement protein expressed in transgenic plants on the permeability of plasmodesmata

“…The amount of P3 in transgenic plants was at least equivalent to that found in systemically infected leaves. It was estimated to be around 100 ng/g of fresh material by quantitative Western blot assay (Berna et al, 1986), using Escherichia coli-produced P3 (Schoumacher et al, 1992a) as standard (data not shown). Controls were represented by plants transformed with non-recombinant vectors.…”

Mapping of the RNA-binding Domain of the Alfalfa Mosaic Virus Movement Protein

“…1 was obtained by site-directed mutagenesis (creation of unique HindIII sites) and segment reassortment. Mutagenesis was carried out on the plasmid designated pSel 1 .P3N (Schoumacher et al, 1992b Fig. 1.…”

“…The coding sequences to be expressed, obtained as N c o I -B a m H I fragments from the pSell .P3N-derived plasmids, were inserted between the NcoI and the B a m H I sites of the expression vector pET3d. The recombinant plasmids were cloned into E. coli strain BL21(DE3) and the proteins were extracted from inclusion bodies of the IPTG-induced bacteria with a urea-containing buffer (Schoumacher et al, 1992b). They were purified by SDS-PAGE in a 10 % gel (Laemmli, 1970), eluted from the gel according to Hager & Burgess (1980), renatured by dialysis against 50 mu-Tris-HC1, pH 7.5, containing 10 mM-2-mercaptoethanol and 1% Tween-20 and stored at -2 0 °C after addition of glycerol (50% v/v final concentration).…”

“…They were purified by SDS-PAGE in a 10 % gel (Laemmli, 1970), eluted from the gel according to Hager & Burgess (1980), renatured by dialysis against 50 mu-Tris-HC1, pH 7.5, containing 10 mM-2-mercaptoethanol and 1% Tween-20 and stored at -2 0 °C after addition of glycerol (50% v/v final concentration). The purified mutant proteins were analysed in parallel with the full-length protein (Schoumacher et al, 1992b) by denaturing electrophoresis (Fig. 2a) and immunoblotting with an anti-P3 serum (Fig.…”