The Involvement of Protein L 11 in the Joining of the 30‐S Initiation Complex to the 50‐S Subunit

Abstract

Ribosomal protein L11 participates in the coupling of the 30-S initiation complex with the 5 0 3 subunit.P3, cores, lacking L7, L8, L12, L33, L10 and L11 were reconstituted with L7 and L10. These particles are unable to join successfully to the 30-S initiation complex, whereas reconstitution of the same cores in the presence of L7, L10 and L l l restores 60-80% of the original coupling activity. Po cores lacking only L7, L8, L12 and L33 are able to carry out one round of initiation, addition of L7 resulting in complete restoration of full activity. The data obtained with these P,, core particles resemble those obtained with untreated 50-S particles carrying thiostrepton, which prevents the binding of initiation factor IF-2 into the 70-S initiation complex.It is postulated that L11 induces a niche on the ribosomal surface to facilitate the proper binding of the IF-2 GTP 3 Met-tRNA complex. This binding of IF-2 enables the 30-S initiation complex to join to the 50-S subunit, because of the associative ability of IF-2. If joining is impaired then both the level of Met-tRNA binding and of the IF-Zmediated GTP hydrolysis is lowered.The physical association of both ribosomal subunits is a prerequisite for a number of reactions which are carried out by initiation and elongation factors and which are strictly ribosome-dependent. One of the most extensively studied processes is the hydrolysis of GTP mediated by the elongation factors EF-Tu and EF-G, in which it is known that several ribosomal proteins are involved (for a review see [l]). In the overall process of GTP hydrolysis L7 and L12 play an important role [2,3] by supplying the binding sites for these factors to the ribosomal surface [4,5] whereas proteins such as L5, L11 and L18 are more involved in the binding and the hydrolysis of GTP Far less has been discovered about the interaction of initiation factors with ribosomal proteins. It has already been established that L7 and L12 are needed for IF-2-mediated hydrolysis of GTP [lo, 111. Because, however, EF-Tu and IF-2 are mutually exclusive in 70-S initiation complexes, one can assume that initiation factors are also using the same or similar set of ribosomal proteins to fulfil their functions [12,13]. In this paper we have focussed our attention on possible roles of ribosomal proteins in the step in the 176, 195-197 (1972).

Functional analysis of the uL11 protein impact on translational machinery

“…However, several structural analyses implicate this protein in interaction with IF2 and RF3, indicating involvement of uL11 in the initiation and termination steps of translation, 15,16 which is also supported by several functional analyses. 17,18 The large ribosomal subunit depleted of this protein is able to bind initiation factor IF2 in vitro but it cannot stimulate GTP hydrolysis required for the subunit joining. 19 Similarly, the RF3-dependent release of RF1 and RF2 is drastically decreased when the uL11 protein is absent on the ribosome during termination.…”

Subunit association defects in Escherichia coli ribosome mutants lacking proteins S20 and L11

“…Furthermore, after crosslinking with diepoxybutane, Baumert et al [9] found L7/L12 crosslinked to the RNA of the heterologous subunit (16s rRNA) more efficiently than to the 23s rRNA while Ohsawa et al [13] found L7/L12 to be among the proteins more strongly protected by the 30s subunits from the chemical modification in situ with the site-specific reagent pyridoxal phosphate. Finally, L11 has been implicated in the joining of the natural 30s initiation complex to the 50s subunit, but it has been suggested that its role is to provide the binding site for the initiation factor IF2-GTP complex [26].…”

“…Furthermore, after crosslinking with diepoxybutane, Baumert et al [9] found L7/L12 crosslinked to the RNA of the heterologous subunit (16s rRNA) more efficiently than to the 23s rRNA while Ohsawa et al [13] found L7/L12 to be among the proteins more strongly protected by the 30s subunits from the chemical modification in situ with the site-specific reagent pyridoxal phosphate. Finally, L11 has been implicated in the joining of the natural 30s initiation complex to the 50s subunit, but it has been suggested that its role is to provide the binding site for the initiation factor IF2-GTP complex [26].In the present paper, we made use of two quantitative tests to study subunit association; one is based on the physical association of the subunits, as measured by a shift of radioactive 30s subunits to faster-sedimenting species in the presence of the 50s subunit, the other on the protection of ribosomebound AcPhe-tRNA against enzymatic hydrolysis by peptidyl-tRNA hydrolase [28]. The latter test requires not only a physical interaction between subunits to take place but also the correct positioning of the peptidyl-tRNA analogue in the ribosomal P-site.…”

“…Gualerzi, Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 73, D-1000 Berlin 33 both subunits. These include translational termination with RFI and the binding and the ribosome-dependent GTPase activity of EF-Tu, EF-G and IF2 [13,[24][25][26][27]. Furthermore, after crosslinking with diepoxybutane, Baumert et al [9] found L7/L12 crosslinked to the RNA of the heterologous subunit (16s rRNA) more efficiently than to the 23s rRNA while Ohsawa et al [13] found L7/L12 to be among the proteins more strongly protected by the 30s subunits from the chemical modification in situ with the site-specific reagent pyridoxal phosphate.…”

Cotranscription of two genes necessary for ribosomal protein L11 methylation (prmA) and pantothenate transport (panF) in Escherichia coli K-12

“…However, the prmA mutant strains were all phenotypically indiscernible from their wild-type parents, and therefore the function, if any, of this energetically costly methylation of protein L1i is unknown. L1i has been implicated in several aspects of ribosome function and assembly, namely, the stringent response in vivo (6, [43][44][45] and in vitro, ribosomal subunit association (21,31,40), the binding domain of the antibiotic thiostrepton (60), ribosomal protein L16 assembly during 50S subunit reconstitution (9,23), protein * Corresponding author.…”