RIBOSOMAL COMPONENTS NEIGHBORING THE CONSERVED 518-533-LOOP OF 16S RIBOSOMAL-RNA IN 30S SUBUNITS

We report the synthesis of a radioactive, photolabile oligodeoxyribonu cleotide probe complementary to 16S rRNA nucleotides 518-526 and its e xploitation in identifying 30S ribosomal subunit components neighborin g its target site in 16S rRNA. Nucleotides 518-526 lie within an almos t universally conserved single-stranded loop that has been linked to t he decoding region of Escherichia coil ribosomes. On photolysis in the presence of activated 30S ribosomes, the probe site-specifically inco rporates into proteins S3, S4, S7, and S12 (identified by SDS-PAGE, RP -HPLC, and immunological analysis); nucleotides C525, C526, and G527 a djacent to its target binding site; and the 3'-terminus of 16S rRNA. W hen the probe is photoincorporated into 30S subunits subjected to brie f cold inactivation (SI subunits), S7 labeling is increased compared t o activated subunit incorporation, while S3, S4, and S12 labeling is d ecreased, as is labeling to nucleotides C525, C526, and (G527; labelin g at the 16S rRNA 3'-terminus appears unchanged. Longer cold inactivat ion of the 30S subunits (LI subunits) leads to decreases in the labeli ng of all components. These results provide clear evidence that C526 l ies within 24 Angstrom (the distance between C526 and the photogenerat ed 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 wi th the probe complementary to 16S rRNA nucleotides 518-526 and with a probe complementary to nucleotides 1397-1405 [Muralikrishna, P., and C ooperman, B. S. (1994) Biochemistry 33, 1392-13981 also provides evide nce for proximity between C526 and G1405. Our results support the conc lusion of Dontsova et al. [Dontsova, O., et al. (1992) EMBO J. 11, 310 5-3116] in placing the 530 loop in close proximity to the decoding cen ter of the 30S subunit but are apparently inconsistent with some prote in-protein distances determined by neutron diffraction [Capel, M. S., et al. (1988) J. Mol. Biol. 200, 65-87]. This inconsistency suggests t hat a multistate model of subunit conformation may be required to acco unt for the totality of results pertaining to the internal structure o f the 30S subunit.