Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins

Abstract

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map ( In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, S11, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 8C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II9 (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics.Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.

Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

“…The most efficient and complete reconstitution reactions occurred with all the proteins added to the rRNA at the same time, which is consistent with previous work showing that protein binding and rRNA folding occur concurrently (17). This is in contrast to observations of reconstitutions with a full complement of recombinant r-proteins, for which an ordered addition of proteins produces a more efficient reconstitution (33). In these prebinding PC/QMS experiments, the absence of certain proteins during the prebinding stage may have led to a less efficient reconstitution by causing a portion of the molecules to form kinetic traps.…”

Assembly of an RNA-Protein Complex

“…coli NusE was purified as described above. Despite previous reports that this protein is insoluble in its native form (24,33), we note that concentrations of NusE up to a concentration of ϳ20 M could be obtained by slow dialysis through a urea gradient (from 6 M to native buffer). Previous data from proteolytic digests and chemical degradation experiments had suggested that free E. coli NusE might exist in a relatively unstructured form (34), and because NusE from Mycobacterium tuberculosis has also been shown (by CD spectroscopy and NMR) to be relatively unstructured (even when bound to NusB (35)), it seems likely that E. coli NusE is also largely unstructured.…”

“…The relative concentrations of ribosomal proteins and ribosomal RNA are modulated by a feedback mechanism that incorporates the ribosomal proteins as components in the antitermination system (13,20). NusE (ribosomal S10 protein) is a small protein that binds specifically to 16 S RNA in the final phase of 30 S subunit assembly (33,43,44), as well as participating as an antitermination factor in rRNA transcription. Therefore, it seems reasonable that NusB determines the specificity of the assembly of the core antitermination complex assembly on boxA RNA sequences (Figs.…”

Assembly of the Archaeal Box C/D sRNP Can Occur via Alternative Pathways and Requires Temperature-facilitated sRNA Remodeling

“…This requirement for elevated temperature is reminiscent of in vitro assembly of Escherichia coli 30 S ribosomal subunits where heating steps during sequential ribosomal protein binding are necessary to facilitate assembly of a functional ribosome subunit. 58 However, protein-induced changes in RNA structure are not limited to elevated temperatures as recently demonstrated with the binding of SRP19 at ambient temperature to alter SRP RNA structure during RNP assembly. 59 Thus, remodeling of RNA structure facilitated by bound proteins appears to be a common and perhaps unifying theme in RNP assembly pathways.…”