Ribosomal Components Neighboring the Conserved 518-533 Loop of 16S rRNA in 30S Subunits

Abstract

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 518-526 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 518-526 lie within an almost universally conserved single-stranded loop that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S ribosomes, the probe site-specifically incorporates into proteins S3, S4, S7, and S12 (identified by SDS-PAGE, RP-HPLC, and immunological analysis); nucleotides C525, C526, and G527 adjacent to its target binding site; and the 3'-terminus of 16S rRNA. When the probe is photoincorporated into 30S subunits subjected to brief cold inactivation (SI subunits), S7 labeling is increased compared to activated subunit incorporation, while S3, S4, and S12 labeling is decreased, as is labeling to nucleotides C525, C526, and G527; labeling at the 16S rRNA 3'-terminus appears unchanged. Longer cold inactivation of the 30S subunits (LI subunits) leads to decreases in the labeling of all components. These results provide clear evidence that C526 lies within 24 A (the distance between C526 and the photogenerated nitrene) of proteins S3, S4, S7, and S12 and the 3'-terminus of 16S rRNA. The identity of the tryptic digestion patterns of S7 labeled with the probe complementary to 16S rRNA nucleotides 518-526 and with a probe complementary to nucleotides 1397-1405 [Muralikrishna, P., & Cooperman, B. S. (1994) Biochemistry 33, 1392-1398] also provides evidence for proximity between C526 and G1405. Our results support the conclusion of Dontsova et al. [Dontsova, O., et al. (1992) EMBO J. 11, 3105-3116] in placing the 530 loop in close proximity to the decoding center of the 30S subunit but are apparently inconsistent with some protein-protein distances determined by neutron diffraction [Capel, M. S., et al. (1988) J. Mol. Biol. 200, 65-87]. This inconsistency suggests that a multistate model of subunit conformation may be required to account for the totality of results pertaining to the internal structure of the 30S subunit.

RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli

“…One possible candidate for such a structure is the 530 stem-loop, one of the most highly conserved regions of the 16S rRNA molecule (44) and one whose secondary structure is influenced by interaction with S12 (45). Cross-linking studies have shown that this loop contacts S12 in E. coli (46), where rimO is present, yet intriguingly, the equivalent loop in 18S rRNA does not appear to contact S23 in humans (47), where rimO is absent. This hypothesis implies that only a stable S12-rRNA complex such as the assembled 30S ribosomal subunit (in which the surface to which S12 is bound is exposed to solution), and not free S12, can serve as an efficient substrate for RimO.…”

Protein synthesis by single ribosomes

“…Escherichia coli Q13 70S ribosomes and 30S and 50S subunits were prepared and activated as described earlier (Alexander et al 1994). ]-30S subunits with a 1.5 M excess of 50S subunits and purifying the labeled 70S ribosomes by fractionation following ultracentrifugation through a linear sucrose concentration gradient (15%-30%).…”

Pokeweed Antiviral Protein Accesses Ribosomes by Binding to L3

“…Experiments designed to reconstitute the minimal ribosomal particle still capable of enzyme activity have established that L3 is essential for maintaining peptidyltransferase activity (15). A photolabile cDNA probe targeted to the central loop of domain V was shown to cross-link to L3 (26). With the use of a photolabile oligodeoxynucleotide probe complimentary to the ␣-sarcin region of E. coli, Muralikrishna et al (11) recently demonstrated the proximity of the ␣-sarcin region to domains IV and V of E. coli rRNA.…”