Distances between 3' ends of ribosomal ribonucleic acids reassembled into Escherichia coli ribosomes

Abstract

The three ribonucleic acids (RNAs) from Escherichia coli ribosomes were isolated and then labeled at their 3' ends by oxidation with periodate followed by reaction with thiosemicarbazides of fluorescein or eosin. Ribosomal subunits reconstituted with the labeled RNAs were active for polyphenylalanine synthesis. The distances between the 3' ends of the RNAs in 70S ribosomes were estimated by nonradiative energy transfer from fluorescein to eosin. The percentage of energy transfer was calculated from the decrease in fluorescence lifetime of fluorescein in the quenched sample compared to the unquenched sample. Fluorescence lifetime was measured in real time by using a mode-locked laser for excitation and a high-speed electrostatic photomultiplier tube for detection of fluorescence. The distances between fluorophores attached to the 3' ends of 16S RNA and 5S RNA or 23S RNA were estimated to be about 55 and 71 A, respectively. The corresponding distance between the 5S RNA and 23S RNA was too large to be measured reliably with the available probes but was estimated to be greater than 65 A. Comparison of the quantum yields of the labeled RNAs free in solution and reconstituted into ribosomal subunits suggests that the 3' end of 16S RNA does not interact appreciably with other ribosomal components and may be in a relatively exposed position, whereas the 3' ends of the 5S RNA and 23S RNA may be buried in the 70S ribosomal subunit.

Structure and function of ribosomal RNA

“…1 and 2), are all obviously directly relevant. In addition, the distances between the 3'-ends of 5S, 16S and 23S RNA in 70S ribosomes are known from energy transfer measurements with fluorescent probes (Odom et al, 1980), and the distances between the 3'-end of 16S RNA and probes attached to proteins SI and S21 have similarly been determined in various functional states (e.g. Odom et al, 1984a).…”

The distance between S1, S21, and the 3′ end of 16S RNA in 30S ribosomal subunits

“…We also report the effect of poly(U) and 50s subunits on these distances. E. coli K12, strain A19, was grown as previously described [25]. A mutant of A19 containing altered and more readily removable protein S21 was isolated by Dabbs [26].…”

“…The mutant, designated VT442, was grown in the medium previously described [26] in a 50-1 fermenter at 35 "C. Harvesting of the cells by the continuous-flow method was begun when the A650 of the culture reached 0.8. Ribosomes were prepared as previously described [25]. The procedure used for separation of subunits has been described in the same reference.…”

“…16s RNA was oxidized with periodate and labeled with fluorescein 5'-thiosemicarbazide (FTS) as previously described [25].…”

“…16s FTS-RNA was reconstituted into 30s particles by the total reconstitution technique essentially as described by Traub and Nomura [31] as modified by Odom et al [25]. The particles were isolated by ultracentrifugation.…”