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

Abstract

The subunit association capacity of 30s and 50s ribosomal subunits from Escherichia coli mutants lacking protein S20 or L11 as well as of 50s subunits depleted of L7/L12 was tested by sucrose gradient centrifugation and by a nitrocellulose filtration method based on the protection from hydrolysis with peptidyl-tRNA hydrolase of ribosome-bound AcPhe-tRNA. It was found that the subunits lacking either S20 or L11 display an altered association capacity, while the 50s subunits lacking L7/L12 have normal association behavior. The association of S20-lacking 30s subunits is quantitatively reduced, especially at low Mg2+ concentrations ( 5 -12 mM), and produces loosely interacting particles which dissociate during sucrose gradient centrifugation. The association of L11 -lacking 50s subunits is quantitatively near-normal at all Mgz + concentrations and produces loosely associating particles only at low Mg2+ concentrations (5-8 mM); the mechanism of their association with 30s subunits, however, or the structure of the resulting 30s -50s couples is altered in such a way as to cause the ejection of an AcPhe-tRNA molecule pre-bound to the 30s subunits in response to poly(U).Although the interaction between ribosomal subunits plays a crucial role in protein synthesis, comparatively little information is available concerning the nature of the ribosomal components involved in subunit association.Both ribosomal RNA and proteins have been located at or near the interface between subunits. The involvement of rRNA in subunit interaction has been demonstrated by chemical and enzymatic modifications of 16s and 23s rRNA in situ and by using specific cDNA probes [l -71. The involvement of ribosomal proteins, on the other hand, is more controversial since data suggesting their direct participation in this process [4, are countered by reports that proteins are absent from the contacting surfaces of the subunits [14].Earlier genetic studies employing different selection methods yielded, amongst several mutants with different altered ribosomal proteins, mutants lacking protein S20 [I 5, 161 and L11 [17]. Protein S20, though formally belonging to the small ribosomal subunit, is occasionally found associated with the large subunit from which it has also been purified and characterized as protein L26 [18, 191. Since S20 seems to partition between 305 and 50s subunits, suggesting a role in subunit interaction, in this paper we investigated the subunit association property of ribosomes derived from a mutant lacking this protein. In addition, we also investigated the association capacity of 50s subunits from a mutant lacking L11 or after the selective removal from the 50s subunit of L7/ L12 [20]. Two dimers of L7/L12 are present in the 50s subunit, probably located in two distinct sites [21, 221. At least one of the two L7/L12 dimers comprises a common domain with L10 and L11 [19, 231 which has been implicated in several ribosomal functions requiring the coordinated presence of Correspondence to C. 0. Gualerzi, Max-Planck-Institut fur Molekular...

The Translation Initiation Functions of IF2: Targets for Thiostrepton Inhibition

“…26,44 Ribosomes lacking ribosomal protein L11 (from E. coli AM68) and protein L11 were prepared as described. 54 Preparation of fMet-tRNA Methionyl-tRNA synthetase (MTS) was overproduced upon IPTG-induction of metG from E. coli JM109 transformed with pNAV7 and partially purified as described. 55 Methionyl-tRNA formyltransferase (FMT) was prepared and partially purified following a published protocol.…”

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.…”

Ribosomes Lacking Protein S20 Are Defective in mRNA Binding and Subunit Association

“…Subunit association is an integral component of translation initiation, and a role for S20 during this step has been previously reported, 5,16 albeit in the absence of supporting kinetic data. In the present study, fast kinetic measurements were used to monitor subunit docking between wild-type 50S subunits and ΔS20 or wild-type 30S subunits.…”

“…15 Followup studies aimed at delineating the ribosomal defect caused by loss of S20 indicated a poor capacity for association with the 50S subunit and a defect at the initiation step. 5,16,17 In one case, the authors suggested poor fMet-tRNA fMet binding and instability of 30S initiation complexes as the sources of defective initiation. 17 In another case, an alteration in the methylation pattern of the 16S rRNA was proposed as the primary cause of defective subunit association.…”