Binding of RrmJ to 50S ribosomal subunits is methyltransferase independent 49

ribosomal subunit depends on its methyltransferase activity and/or AdoMet binding, the binding of two methyltransferase deficient RrmJ mutants was tested (Hager et al., 2004). The RrmJ-K38A mutant protein harbors a mutation in the active site of RrmJ rendering RrmJ methyltransferase inactive, while the RrmJ-D83A mutant protein has a mutation in the AdoMet binding site, which makes the enzyme unable to bind the cofactor. The two inactive RrmJ mutant proteins were incubated with 50S ribosomal subunits prepared from the rrmJ deletion strain HB23 in a 2:1 ratio for 10 min at 37°C and the binding of RrmJ was analyzed on sucrose gradients. As shown in Fig. 21, both mutants were able to bind to the 50S ribosomal subunit with the same apparent affinity than wild type RrmJ. These results clearly emphasized the fact that the binding ability of RrmJ to the 50S ribosomal subunit is not dependent on its methyltransferase activity. Furthermore, since the 50S ribosomal subunits used in this experiment were prepared from the rrmJ deletion strain and, therefore, do not harbor the methylated U2552, it also shows that RrmJ’s binding does not depend on any conformational A-loop rearrangements that might be caused by the Um2552 modification (Blanchard and Puglisi, 2001).

Figure 21: RrmJ’s binding is methyltransferase activity independent

50S ribosomal subunits prepared from an rrmJ deletion strain were incubated with the inactive mutant K38A and the AdoMet binding deficient mutant D83A in a 1:2 ratio. After applying onto 10-50% sucrose gradients, fractions were collected and analyzed by western blot.

2.6.4 Determination of RrmJ’s binding site

RrmJ was found to stay associated with the 50S ribosomal subunit. Therefore it was interesting to investigate where the binding site for RrmJ is located on the surface of the 50S ribosomal subunit. Due to its methylation target, the A-loop of the 23S rRNA, RrmJ presumably binds on the intersubunit face of the 50S ribosomal subunit close to the peptidyltransferase center. In order to pinpoint its exact location crosslinking studies were performed to identify interactions between RrmJ and ribosomal proteins. The attempt was to crosslink RrmJ to proteins of the 50S ribosomal subunits prepared from the rrmJ deletion strain using the chemical crosslinker glutaraldehyde (GA). In the presence increasing of concentrations of glutaraldehyde RrmJ was incubated with 50S ribosomal subunits at 37 ºC for 2 min. Then, SDS-PAGE and western blot analysis was used to detect RrmJ. In the crosslinking experiment two additional bands appeared that were not visible when RrmJ was incubated with GA in the absence of 50S ribosomal subunits (Fig. 22). This suggested that these two bands represent complexes between RrmJ and a ribosomal protein. Since both bands became only apparent when GA was used in a concentration of 600 µM or higher, this GA concentration was used for further optimization attempts. The same two bands became visible when 50S ribosomal subunits from the rrmJ wild type strain were supplemented with exogenous RrmJ and used for crosslinking studies. In order to see the crosslinked complexes on a colloidal coomassie blue stained SDS-PAGE, which would enable us to analyze the protein complexes by mass-spectrometry, various attempts were made to upscale the incubation reaction. Unfortunately, I was never able to see any cross-linked bands in quantities that were large enough to identify by mass-spectrometry.

Figure 22: Crosslinking of RrmJ to the 50S ribosomal subunit

200 nM RrmJ was incubated with or without 200 nM 50S ribosomal subunits prepared from the rrmJ deletion strain in the presence of increasing concentrations of glutaraldehyde (GA) at 37ºC for 2 min.

The reaction was stopped by adding Tris, pH 8.0, to a final concentration of 20 mM. The samples were loaded onto a 14% SDS PAGE and western blot analysis was performed with polyclonal antibodies against RrmJ.

Lane 1 shows purified wild type RrmJ as standard. Lanes 2-7 show RrmJ crosslinked to 50S ribosomal subunits using 20, 100, 250, 400, 600 and 800 µM GA, respectively. Lanes 8 – 13 show RrmJ alone crosslinked with 20, 100, 250, 400, 600 and 800 µM GA, respectively. The arrows point to the bands that appeared only when RrmJ is cross-linked to 50S ribosomal subunit reactions, but not in the crosslinking reactions with RrmJ alone, therefore showing protein complexes consisting of RrmJ and ribosomal protein. 600 µM GA appears to be the optimal concentration for obtaining both crosslinked complexes.

2.6.5 30S ribosomal subunits displace RrmJ during ribosome assembly

Under destabilizing “dissociating” salt conditions, where 70S ribosomes rapidly dissociate into their ribosomal subunits, all of the endogenous RrmJ was found to be stably associated with the 50S ribosomal subunits. In contrast, however, under associating salt conditions where the 70S ribosomes stay intact, most of RrmJ was found free in the “top of the gradient” (compare Fig. 20A and B). This suggested that assembly of the 70S ribosome might cause the displacement of RrmJ from the 50S ribosomal subunit. Considering the fact that RrmJ’s methylation target is the A-loop of the 23S rRNA, which is located directly at the inter-subunit space, it appeared plausible that the displacement of RrmJ was caused by the assembly of 30S and 50S. To investigate this in more detail, in vitro ribosome reconstitution experiments were performed according to Nierhaus et al. (Blaha et al., 2002). 50S ribosomal subunits prepared from the rrmJ wild type strain were supplemented with additional purified RrmJ and the RrmJ-50S ribosomal subunit complexes were incubated with 30S ribosomal subunits in a 1:2 ratio. Then, the ribosome reconstitution was analyzed on sucrose gradients

and the localization of RrmJ was monitored. To avoid nonspecific interaction of RrmJ with the 30S subunits, salt concentrations had to be used that were slightly higher (100 mM KCl) than the reported salt conditions (30 mM KCl) that allow the reconstitution of 30S and 50S into 70S (Blaha et al., 2002). Under these chosen salt concentrations, association of 30S and 50S ribosomal subunits do not produce 70S particles but generate a ~ 55S ribosomal particle instead (Blaha et al., 2002). As shown in Fig. 23 (dashed line), incubation of isolated 30S and 50S ribosomal subunits under conditions that do not support ribosome assembly, did not cause the dissociation of 50S associated RrmJ. In contrast, however, incubation at 40^oC, which triggers ribosome assembly in vitro, quickly led to the formation of the ~55S particle and the almost complete dissociation of RrmJ (Fig. 23, dotted line). This finding clearly indicated that RrmJ is released from the 50S ribosomal subunit in the course of ribosome assembly.

Figure 23: 30S ribosomal subunits replace RrmJ during ribosome assembly

30S and 50S prepared from the rrmJ wild type strain (HB24) were incubated in reconstitution buffer (20mM Hepes-KOH, pH 7.5, 20 mM MgCl₂, 100 mM KCl, 4 mM β-mercaptoethanol, 40 U of RNasIn) in the presence of 50µM AdoMet and exogenous wild type RrmJ in stoichiometric amounts to 50S at 40ºC for 10 min (dotted line) or left on ice (dashed line). Subsequently, the samples were analyzed on 10-50 % sucrose gradients. Continuous fractions were collected and western blot analysis was performed.