Folding of precursor subunits and assembly of r-proteins

The in vitro reconstitution of translational active ribosomal subunits from naked rRNA and purified components was one of the biggest breakthroughs in understanding how cells produce ribosomes (Hosokawa et al., 1966; Traub and Nomura, 1968; Nomura and Erdmann, 1970). Although the assembly and folding process of the large ribosomal subunit is more complex and probably involves many intermediates, the general principles of assembly and folding of both subunits are the same (Nomura et al., 1970; Herold et al., 1987). These will be elucidated hereafter, based on the excessive studies of SSU folding and assembly (reviewed in (Woodson, 2008; Sykes et al., 2009)).

The in vitro studies showed that the ribosome is a self-assembling RNP, since reconstitution of both subunits required no additional factors. In addition, the primary sequence of the 16S itself, in particular of the 5'-domain was sufficient to form many of the interactions observed in the three-dimensional structures (Stern et al., 1989; Adilakshmi et al., 2005). Nevertheless, thermodynamically traps of rRNA folding are greatly reduced upon r-protein binding (Semrad et al., 2004; Woodson, 2008) and in vivo most probably also by biogenesis factors (among others (El Hage et al., 2001; Maki and Culver, 2005; Hoffmann et al., 2010; Bunner, Nord, et al., 2010a)).

Introduction

Figure 16. The Nomura assembly map for the 30S subunit

The assembly map shows the order of r-proteins in respect of their hierarchy of binding to the different subdomains of 30S subunits. Primary binders are indicated with 1°, secondary binders with 2° and tertiary binders with 3°. For a projection into 3D see Figure 5. (modified from Sykes and Williamson, 2009)

Another observation during the reconstitution experiments was that assembly of r-proteins seems to follow a hierarchy. In other words, the binding of some r-proteins is the prerequisite for stable incorporation of others. The r-proteins were grouped into three categories: primary binders – required for initial folding of rRNA, bind first; secondary binders – stable incorporation depends on the preceding incorporation of primary binders;

tertiary binders – largely depend on primary and secondary binders (Figure 16).

Strikingly, the hierarchical assembly of the SSU subdomains (see 1.2.2) is, with minor exceptions, independent of each other (Figure 16). The electron micrographs in Figure 17 nicely illustrate the interplay of 16S rRNA folding and r-protein assembly

Figure 17. Electron micrographs taken during the in vitro assembly process of E. coli SSUs

30Å EM images taken during the in vitro reconstitution of the E. coli 30S ribosomal subunit. (a) free 16S rRNA. (b) 16S rRNA and primary binding r-proteins (16S rRNA and S4, S8, S15, S20, S17, S7). (c)-(g) Subsequent assembly of the missing r-proteins. (h) and (i) fully in vitro assembled SSU (16S rRNA and 20 r-proteins or 16S rRNA and TP30, respectively). (j) native E. coli SSU. (modified from Mandiyan et al., 1991)

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(Mandiyan et al., 1991). See particularly Figure 17 (d), in which all three subdomains are already distinguishable by assembly of the primary (S4, S8, S15, S20, S16/17, S7) and secondary (S16, S19, S13, S9, S18, S6) binding r-proteins. Recent work showed that this autonomous subdomain folding simultaneously starts at different sites all over the 16S rRNA and is generally clustered in the SSU subdomains (Talkington et al., 2005; Adilakshmi et al., 2008). It has even been possible to almost fully reconstitute the SSU subdomains independently, starting from 16S rRNA fragments and the respective r-proteins (Weitzmann et al., 1993; Samaha et al., 1994; Agalarov et al., 1998).

The binding of r-proteins apparently follows most likely an induced fit model (Adilakshmi et al., 2008). The first interaction of one r-protein part induces re-folding of the local rRNA environment, which in turn creates the binding site for another protein part or another r-protein. By this mechanism it is possible that r-proteins of different levels in the assembly hierarchy bind cooperatively. They maybe initially contact rRNA simultaneously, but stable binding of secondary or tertiary binders through induced fit requires structural re-arrangement by the primary binder.

The results of the in vitro dependency map (Mizushima et al., 1970), pulse-chase experiments with labeled r-proteins during reconstitution (Talkington et al., 2005; Bunner, Beck, et al., 2010) and time-resolved hydroxyl radical footprinting (Adilakshmi et al., 2008) all lead to the following two conclusions: first, the hierarchy of r-protein binding correlates mostly with the temporal order of assembly and second, the 5'-domain is faster decorated with r-proteins than the central or 3'-domain. In addition, the transcription of rDNA genes in vivo and the assembly of r-proteins most probably is coupled (Chooi and Leiby, 1981; Gallagher et al., 2004; Koš et al., 2010). Transcription is 5' to 3' directed and the binding sites of many primary binders are at the 5'-ends of 16S and 23S rRNA. These observations led to the postulation of the so-called “assembly gradient” (Nierhaus, 1991).

This hypothesis is nevertheless challenged by the facts that nucleation of folding simultaneously starts at many different sites (Adilakshmi et al., 2008) and initial binding of a subset of r-proteins (5'-, and central domain and/or primary binder of the 3'-domains) does not enhance the assembly of other 3'-domain binding proteins (Bunner, Beck, et al., 2010).

Interestingly, the wildtype-like array of the subdomains of each subunit in the rRNA operons is not essential in vivo. E. coli strains, in which the subdomains were permuted, were able to grow and exhibited fully assembled ribosomal subunits (Kitahara and Suzuki, 2009).

The knowledge about the hierarchy of r-protein assembly in eukaryotes is rather limited. Two systematic knockout screen in S. cerevisiae showed that r-proteins that bind in the 5'- and central domain of the small subunit were required for early processing steps (A0, A1, A2) and proteins of the head domain were required for efficient D site cut processing (Ferreira-Cerca et al., 2005). This clear clustering is however not true for r-proteins of the large ribosomal subunit (Pöll et al., 2009). A detailed in vivo analysis of head domain assembly in yeast

Introduction

demonstrated a broad homology between the eukaryotic and bacterial r-protein assembly pathway in this SSU subdomain (Ferreira-Cerca et al., 2007). Depletion of the primary binder of the head domain rpS5 (S7 in Bacteria) consequently led to loss of stable binding of other head domain r-proteins, while the 5'- and central domains were normally assembled. The second level in the hierarchy of assembly might be as well conserved, since depletion of rpS15 (S19 in Bacteria) led to loss of stable binding of only a smaller subset of r-proteins.

Although many of the r-proteins are able to associate already with nascent SSUs in the nucleus (among others (Ferreira-Cerca et al., 2005, 2007; Krüger et al., 2007)), their final, stable incorporation could be dependent on cytoplasmic maturation events (see before:

induced fit model). One well studied example is rpS3, whose stable incorporation into nascent SSU depends on a phosphorylation/dephosphorylation cycle in which the cytoplasmic kinase Hrr25p is involved (Schäfer et al., 2006). Other examples are rpL10, rpL24 and the phospho-stalk protein P0, all incorporated into pre-60S subunits after biogenesis factor displacement in the cytoplasm (among others (West et al., 2005; Pertschy et al., 2007; Kemmler et al., 2009)). Early work in the late 1970s identified potential late binding r-proteins by comparison of the incorporation of labeled r-proteins into ribosomal subunits after short or long pulse times (Kruiswijk et al., 1978; Auger-Buendia et al., 1979).

They found rpS10, rpS25, rpS27, rpS31, rpS32, rpS33 and rpS34 respectively rpS7, rpS9, rpS20 and rpS26 to associate with nascent subunits at a late stage of ribosome assembly.

The major difficulty here is the assignment of the ribosomal proteins (see 1.2.1) according to the particular migration behavior in the 2D gels (McConkey et al., 1979). Each laboratory used its own protocol for the gel-electrophoresis, so e.g. rpS33 could be rpS28, rpS9 could be rpS10, and so forth. Taken together, these data imply nevertheless a correlation of the rRNA processing phenotype upon depletion of the r-protein and its stage of stable assembly (see before: the “assembly gradient”).

In all evolutionary kingdoms a class of biogenesis factors exists that is thought to improve/accelerate the assembly of r-proteins onto nascent subunits. A recent study used labeled protein pulse–chase experiments monitored by quantitative mass spectrometry, to analyze the effects of these assembly factors on in vitro reconstitution kinetics of bacterial 30S subunits (Bunner et al., 2010b). They concluded that assembly factors facilitate the binding of r-proteins through induced conformational changes, RNA chaperone-like activity or inhibition of unproductive r-protein assembly by physically blocking the binding site. In eukaryotes, the phenotypes observed upon depletion of some r-protein assembly factors can be (partially) suppressed by overexpression of the respective r-protein (among others (Baudin-Baillieu et al., 1997a; Loar et al., 2004; Buchhaupt et al., 2006)). These assembly factors are therefore not utterly required for r-protein binding, but they accelerate stable incorporation and thereby ribosome biogenesis.

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