Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Abstract

Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent significant conformational changes that were governed by the cooperative interactions of the ribosomal proteins. The sequential association of the first 12 proteins with the 16S rRNA resulted in the formation of core particles containing up to three mass centers at distinct stages of the assembly process. These globular mass centers may correspond to the three major domains (5', central, and 3') of the 16S rRNA. Through the subsequent interactions of the late assembly proteins with the 16S rRNA, two of the three domains merge, yielding the basic structural traits ofthe native 30S subunit. The fine morphological features of the native 30S subunit became distinctly resolved only after the addition of the full complement of proteins. The fully reconstituted 30S subunits are active in polyphenylalanine synthesis assays. Visualiztion of the assembly mechanism of the E. coli 30S ribosomal subunit revealed domain-specific folding of the 16S rRNA through the formation bf distinct intermediate core particles hitherto not observed.Ribosomes are ubiquitous macromolecular assemblies of proteins and ribonucleic acids involved in protein biosynthesis. This bipartite cellular organelle is composed of a large and a small subunit. The structure-function relationship of the small (30S) ribosomal subunit ofEscherichia coli has been extensively studied. This subunit is actively involved in the initiation of protein synthesis by its interactions with initiation factors, messenger RNAs, transfer RNAs, and the large (50S) ribosomal subunit (1-4). The 30S subunit possesses characteristic structural features known as the head, body, cleft, and platform (5, 6) and is composed of a single 16S rRNA molecule and 21 different ribosomal proteins (S1-S21). Several ribosomal proteins interact with the 16S rRNA through a sequential and cooperative process as revealed from the in vitro assembly of the 30S subunit (7-9). Results obtained from these studies led to the classification of the proteins as primary binding proteins, which bind directly to the 16S rRNA, and secondary binding proteins, which require the previous association of the primary binding proteins. Apart from being the structural base for protein interactions, the 16S rRNA has an established role in protein biosynthesis (10-14). However, the role of the ribosomal proteins in ribosome structure and function remains unclear.Scanning transmission electron microscopy (STEM) was used to quantitatively evaluate the conformational changes induced in the 16S rRNA molecule by its interactions with the ribosomal proteins under nondenaturing conditions (15). Unlike other physical techniques tha...

The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli

“…Sequential and cooperative interaction between the HSP70 (DnaK) and HSP60 (GroEL) to promote protein folding (Gragerov et al 1992;Langer et al 1992;Ziemienowicz et al 1993;Gaitanaris et al 1994;Petit et al 1994;Buchberger et al 1996;Thomas and Baneyx 1996;Dionisi et al 1998;Nishihara et al 1998), protein export (Phillips and Silhavy 1990), and protein disaggregation (Kedzierska et al 1999) has been well documented. An alternative possibility is that chaperones contribute to ribosome assembly by virtue of their interaction with ribosomal RNA: During ribosome biogenesis, rRNAs are subject to post-transcriptional modi®cations, conformational changes (Mandiyan et al 1989(Mandiyan et al , 1991 and trimming. The eukaryotic HSP70, HSP110 (Henics et al 1999) and HSP101 (Wells et al 1998 ) have been reported to have the capacity to bind RNA, and this could be relevant for the late events of ribosome assembly (Weeks 1997).…”

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

“…The striking similarity of phenotype between rpl23aa, rps5b, rps13b, rps18a, rpl24b, and Atnuc-l1 mutants may thus represent the in vivo response to decreased production of processed rRNAs and the concomitant effects on ribosome biogenesis and protein production, with the observed severity gradient being a measure of the extent of the reduction in mature rRNAs. In this scenario, r-proteins likely act indirectly on pre-rRNA processing, with their binding required to induce conformational changes that facilitate pre-rRNA processing or allow the association of other r-proteins and factors involved in processing (Mandiyan et al, 1991;Ban et al, 2000;Wimberly et al, 2000;van Beekvelt et al, 2001).…”

Chloroplast Ribosomal Protein S7 of Chlamydomonas Binds to Chloroplast mRNA Leader Sequences and May Be Involved in Translation Initiation

“…These interactions have been well defined and are highly conserved throughout the prokaryotes (Nowotny and Nierhaus, 1988;Chiaruttini et al, 1989;Mandiyan et al, 1991;Dragon and Brakier-Gingras, 1993;Samaha et al, 1994;Spiridonova et al, 1998;Miyamoto et al, 1999). The S7 protein has been crystallized from Thermus thermophilus (Wimberly et al, 1997) and Bacillus stearothermophilus (Hosaka et al, 1997), and the active site structures needed for formation of the ribosomal small subunit and to bind the rpsG mRNA 5ЈUTR (rpsG is the functional equivalent of rps7 in Chlamydomonas chloroplasts) have been determined at a resolution of 1.9 Å (T. thermophilus) and 2.5 Å (B. stearothermophilus).…”