In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

Abstract

In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, SlO, S13, S14, S16, S19, and S20. (4), and Schweiger and Gold (5). In preliminary experiments we obtained better results using the former system (4, 6); we therefore decided to modify that system. Our major modification was to fractionate the "S30" of the Zubay system into salt-washed ribosomes, an initiation factor fraction ("IF"), a partially purified "enzyme fraction" ("SlOODE"), and DNA.-dependent RNA polymerase. S1OODE contains EF-T, EF-G, and aminoacyl-tRNA synthetases, and is devoid of nucleic acids. Amino-acid incorporation in the absence of added DNA is very small (see [35S ]Methionine was incorporated into protein with E. coli DNA as a template, essentially as described in Table 1. After 2 hr of incubation, the reaction mixtures were cooled and dialyzed against TRI buffer containing 8 M urea and then TMAI buffer containing 8 M urea. This treatment dissociated the ribosomes in the incubation mixture so that the released r-proteins could mix with radioactive r-proteins synthesized in the system. The resultant solution was passed through a DEAE-cellulose column (1 X 10 cm, equilibrated with 8 M urea in TMAI buffer, pH 7.4). Most of the 30S r-proteins lassed through the column. However, the "acidic" r-proteins, S1, S2, and S6, were probably retained together with other "soluble proteins" and nucleic acids (9). The pass-through fraction ("basic protein fraction") contained about 50% of the total proteins.Carrier nonradioactive 30S r-protein mixture (TP30) and 16S RNA were then added to the basic protein fraction, and 30S ribosome reconstitution was performed (7,8

Specialized transducing phages for ribosomal protein genes of Escherichia coli.

“…Those proteins where there is significant overlap include: S9 and S11, S5 and Lii, L8 and L9, L14 and L15, and S15 and S17. Proteins L26 and S20 are the same protein (24 r-proteins were recovered by reconstitution into 30S particles (19). As shown in Fig.…”

“…The identity of the radioactive r-proteins in the reconstituted particles was determined immunologically with antisera to purified 30S proteins as previously described (19). Examples are shown in Fig.…”

“…The [35S]Methionine-labeled protein was synthesized in a DNA-dependent cell-free system, using E. coli DNA [prepared as described previously (19)], Xdspcl, or XCI-857S7 DNA [prepared as described by Zubay, et al (25)] as template. Radioactive 30S r-proteins were purified by passing the in vitro products through a DEAE-cellulose column and reconstituting 30S ribosomal subunits in the presence of carrier nonradioactive 16S RNA and 30S r-proteins (19). After reconstitution the samples were centrifuged through a 5-20% sucrose gradient in 30 mM Tris-HC1 (pH 7.4), 20 mM MgC12, and 0.33 AM KCl using a Spinco SW 27 rotor.…”

“…In addition these phages carry several genes which (Fig. 2) and tested by radioimmunodiffusion assay (19). Each set of pictures shows a gel (left) and an autoradiogram of the gel (right).…”

Effects of ribosomal mutations on the read-through of a chain termination signal: studies on the synthesis of bacteriophage lambda O gene protein in vitro.

“…MATERIALS AND METHODS Phages AcI857S7 (called "A" in this paper), AcI857cII-am60am41 ("AcIIam") (5), Ac8570am125 ("AOaml25"), and AcIam6Oam29 ("AOam29") (6) were used in this work. DNA-dependent protein synthesis in vitro as well as analysis of products was performed as described previously (7,8), expect for the source of ribosomes. Ribosomes were prepared from E.…”