Directed hydroxyl radical probing of 16S ribosomal RNA in ribosomes containing Fe(II) tethered to ribosomal protein S20

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

“…The ability to reconstitute the E. coli 30S subunit in vitro from purified components has allowed detailed investigation of the structure, function, and assembly of the 30S subunit+ However, isolation of sufficient amounts of highly purified, functional small subunit ribosomal proteins from isolated subunits can be difficult, laborious, and costly+ In particular, it is difficult to exclude cross-contamination between ribosomal proteins in large-scale purification+ We sought to alleviate some of these problems by cloning and overexpressing a complete set of recombinant small subunit ribosomal proteins for use in studying the E. coli 30S ribosomal subunit+ Initial attempts at reconstituting 30S subunits with recombinant proteins and isolated 16S rRNA, following standard procedures (Traub & Nomura, 1969), led to very inefficient production of 30S subunits+ Therefore, we developed an ordered assembly protocol that allowed efficient reconstitution of 30S subunits using the purified recombinant proteins+ Reconstitution of 30S subunits with the recombinant proteins is less efficient than those using a complete mixture of ribosomal proteins (TP30) but more efficient than 30S subunit reconstitution using proteins individually purified from ribosomal subunits+ The molecular composition and sedimentation properties of the recombinant 30S subunits are similar to those of natural 30S subunits and those reconstituted with TP30+ Also, the recombinant 30S subunits were active, as measured by in vitro assays+ The large amounts of pure ribosomal proteins that can be obtained greatly facilitate studies that depend on reconstitution of 30S subunits from individual proteins, such as directed hydroxyl radical probing from single positions on individual proteins (Culver & Noller, 1998;Culver et al+, 1999)+ Moreover, the potential for exhaustive mutational analysis of each small subunit ribosomal protein provides a new approach to investigation of their roles in structure, function, and assembly of ribosomes+…”

Assembly of the 5′ and 3′ Minor Domains of 16S Ribosomal RNA as Monitored by Tethered Probing from Ribosomal Protein S20

“…1c) in the 30S subunit have been published. 2 In fully formed 30S subunits, cleavages of the 5′ and 3′ minor domains of 16S rRNA were observed. Consequently, the results of the directed hydroxyl radical probing experiments from r-protein S20 in more minimal complexes will make possible a better understanding of the interactions of the 5′ and 3′ minor domains with this protein.…”

“…S20 is one of a few small subunit ribosomal proteins (r-proteins) that interacts with two domains of 16S ribosomal RNA (rRNA), the 5′ and 3′ minor domains [1][2][3][4] (Fig. 1a and b).…”

“…These studies also allow the cleavage data from Fe(II)-S20 to be examined in the context of the 30S subunit structure. 2,4 The results from this work can be grouped into three main categories. First, many of the cleavage sites, especially those in the 5′ domain, are the same in the binary complex as in the fully assembled particle, although there can be differences in intensity at positions where cleavage is observed in both complexes.…”

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

“…8 High-resolution structural studies have placed S20 at the bottom of the body of the 30S subunit, where it interacts with several helices of the 16S rRNA, including helix 44 (h44). [9][10][11] h44, also known as the penultimate stem, constitutes a major RNA structural element as it runs along the entire length of the 30S body on the intersubunit face. 9 This dynamic helix not only forms many of the bridges connecting the 30S to the 50S subunit in the 70S ribosome but also has been implicated in decoding at the A-site.…”