Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes.

Abstract

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction-namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.The majority ofthe ribosomal proteins in both the small and the large ribosomal subunit bind in vitro to their respective rRNA. In the small subunit, 12 out of21 proteins have been identified as "RNA-binding proteins" (1,2), and in the large subunit, 17 out of 32 (3, 4). Proteins derived from the large ribosomal subunit are designated L.In the case ofthe small ribosomal subunit, it has been shown that the cooperativity of the assembly process is so high that certainly not all 12 but at most 2 or 3 proteins (or protein complexes) can bind independently to 16S rRNA (5).In the case of the large (50S) subunit, it is not yet clear whether even the seven most strongly binding proteins (4) bind independently to their rRNA in the course of the assembly. If this were so, then these proteins would be distributed independently of each other over the various individual rRNA molecules. If the rRNA were present in excess, this random distribution would disturb the formation of ribosomal subunits with a constant and stoichiometric protein content and, as a result, dramatically decrease the output ofactive ribosomes. In fact, a synthesis of rRNA in a molar excess of more than threefold over ribosomal proteinst has been observed under unfavorable growth conditions (6). Therefore, one would expect that, in spite ofthe large number of23S rRNA-binding proteins concerned in the assembly ofthe 50S subunit, only a few ofthese proteins should be able to bind independently to the rRNA (i. e., without cooperativity), thus selecting the rRNA molecules for the 50S assembly in the presence of an rRNA excess. These proteins we term "assembly initiator proteins".In this paper we demonstrate that the in vitro assembly of the large ribosomal subunit is initiated by only two proteins.Two different methods independently identify these proteins as L24 and L3. MATERIAL AND METHODSRibosomes and their subunits were isolated from Escherichia coli cells as described (...

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

“…4B) suggest that L24 folds the fragment by bringing together the loops around 310 and 330. This view agrees with both the phylogenetically proposed pseudoknot (39) and the role of L24 as an assembly initiator protein and organizer of rRNA folding (34,35). L4 interacts mainly with the bulge between helices H19 and helix H20 around U321.…”

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

“…The present analysis strikingly reveals that the RNA adopts a structure in the ternary complex L24-rRNA-L4 that is different from those seen in either binary complex+ Considering the prominent role that both proteins play in early assembly events, the identified short RNA containing the nonoverlapping binding sites of both proteins might be a key element for the 23S rRNA folding during early assembly: L24 is one of the two assembly initiator proteins that bind to the 23S rRNA without the help of other proteins (Nowotny & Nierhaus, 1982)+ L4 together with L24 belongs to the group of five proteins that are essential and sufficient for the formation of the first assembly intermediate during in vitro assembly of the 50S subunit (Spillmann et al+, 1977)+ This assembly intermediate cannot be bypassed during the formation of active 50S particles (Dohme & Nierhaus, 1976)+ L24 has no other role, for example, in the functions of the ribosome, because it can be removed from the ribosome after achieving the early assembly intermediate without affecting the further assembly of active 50S subunits (Spillmann & Nierhaus, 1978)+ This interpretation of in vitro reconstitution experiments is confirmed by in vivo experiments: 23S rRNA deletion variants that lack the rRNA L4 region are not incorporated into mature ribosomal particles (Skinner et al+, 1985;Liiv et al+, 1996), probably because a productive interaction with L24 and L4 is prevented+…”

“…The effects of phosphorothioate modification on RNAprotein interaction match direct structural information obtained from corresponding X-ray structures or that derived by cryoelectron microscopy very well+ For example, results obtained with the MS2 coat protein-RNA (Milligan & Uhlenbeck, 1989;Dertinger et al+, 2000) and tRNA-synthetase systems (Schatz et al+, 1991;Rudinger et al+, 1992;Voertler et al+, 1998) are directly comparable to interactions seen in three-dimensional crystal structures of the complexes+ Phosphorothioated tRNAs bound by the ribosome give specific cleavage patterns that are characteristic for the particular ribosomal binding site (Dabrowski et al+, 1995) and that are dependent on the functional state of the ribosome (Dabrowski et al+, 1998)+ The protection pattern observed with P-site bound peptidyl-tRNA analogs was in perfect agreement with the ribosome contacts seen with an f-Met-tRNA at the ribosomal P site in cryoelectron microscopy pictures (Malhotra et al+, 1998)+ Recently, we selected a short fragment of domain I of 23S rRNA (GG295-343CC, 53 nt) that binds independently and simultaneously the ribosomal proteins L4 and L24 (Stelzl et al+, 2000a(Stelzl et al+, , 2000b, two key proteins for the early assembly of the large subunit of bacterial ribosomes (Nierhaus, 1991)+ L24 is one of the two assembly initiator proteins of the 50S subunit (Nowotny & Nierhaus, 1982), and L4 is essential for the formation of the first assembly intermediate particle (Spillmann et al+, 1977)+ Here, we present phosphorothioate probing data of the ternary L24-rRNA-L4 complex determined by an in-gel analysis and compare them with that of the two binary complexes L24-rRNA and rRNA-L4+ In-gel iodine cleavage enables the probing of RNA structures in homogeneous populations of the different complexes, thereby overcoming the problem that a preparation of a ternary complex still contains a fraction of binary complexes obscuring the signals+ The results demonstrate that the rRNA region within the binary complexes adopts a conformation that is different from that observed in the ternary complex+ We discuss these probing data in light of the recent crystallographically determined three-dimensional model of the 50S ribosomal subunit (Ban et al+, 2000)+…”

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

“…These sequences might have to be juxtaposed during an early compaction event to commence 60S assembly, similar to bacterial large subunit assembly where sequences flanking 23S rRNA form a helix recognized by RNase III (Shajani et al 2011). The bacterial homolog of L3 occupies a similar position close to the ends of 23S rRNA and is required to initiate 50S assembly (Nowotny and Nierhaus 1982).…”