RNA—protein interactions in the ribosome. Binding of proteins L1, L3, L6, L13 and L23 to specific fragments of the 23S RNA

Abstract

Ten of the 33 proteins found in the 50 S subunit of the Escherichia coli ribosome associate independently and specifically to the 23 S ribosomal RNA [l]. The location of the binding sites for these proteins has been studied in two different ways. Branlant et al. [2,3] have isolated ribonucleoprotein particles resulting from extensive nuclease digestion of complexes between proteins Ll, L20, L23, L24 and the 23 S RNA. Sequence analysis of the RNA regions protected from digestion by the proteins enabled these authors to locate the binding sites on the 23 S RNA molecule. On the other hand, Spierer et al. [4] have tested the association of each of the ten binding proteins with two segments of the 23 S RNA produced by limited ribonuclease digestion of the 50 S subunit. One segment sediments at 13 S, covers 40% of the 23 S RNA sequence, and derives from the 5' end of the molecule. It contains the specific binding sites for proteins LA, L20 and L24. The other segment, which sediments at 18 S, encompasses about 60% of the 23 S RNA sequence and arises from the 3' end of the molecule. It contains the specific binding sites for proteins Ll, L2, L3, L6, L13, L16 and L23. We report here the preparation of fragments of the 18 S RNA segment, and the distribution of binding sites for proteins Ll, L.3, L6, L13 and L23 on these fragments

Elongation factor G protects a nuclease‐sensitive site of 23 S RNA within the ribosome

“…It is known that proteins L7/L12, LlO, Lll, L14 and L6 are located in or near this center, can be crosslinked with EF-G and are nearest neighbours (e.g., [lo-14, 35-371). At the same time they bind to remote regions of the 23 S RNA sequence: proteins L7/L12 + LlO and Lll bind to the 5'-terminal 13 S fragment [38,39], protein L14 to the central 8 S fragment [28] and protein L6 to the 3 ' -terminal 11 S (12 S) fragment [33,40]. Besides, according to our data, EF-G is photoaffinity crosslinked with the 3 ' -terminal 18 S fragment and protects site 2 within this fragment from the nuclease whereas diepoxybutane treatment cross-links the factor with the 5 '-terminal 13 S fragment near site 1 (sequence 1055-1081) [34].…”

“…As known, 23 S RNA within the 50 S subparticle of E. coli ribosomes apparently has 3 exposed sites which are most sensitiye to the action of different ribonucleases specific to single-stranded regions of the polynucleotide chain (pancreatic RNase [21][22][23][24][25][26][27][28][29], TI-RNase [30] and endogenous ribosomal RNase from E. co& [26]). One of these sites (site 1, see scheme in fig.1) is positioned at a distance of about 1171-l 178 nucleotides from the 5 '-end of 23 S RNA [31,32] and its hydrolysis gives two fragments detected by SDS-PAGE without denaturation of the RNA secondary structure: the 3 '-terminal 18 S and 5 '-terminal 13 S fragments [26-281. Electrophoresis of the hydrolyzate after preliminary denaturation of the RNA (5 min at 75-8O"C, 0.2% SDS [24] or 6.6-8.0 M urea [25,30]) reveals another specific cleavage site (site 2 in the scheme) at a distance of about 1000 nucleotides from the 3'-end of 23 S RNA with formation of the 3'-terminal 11 S [28,32) (or I2 S [30,33]) fragment. The third cleavage site (site 3 in the scheme) detected also on-Iy in a denatured sample is located somewhere within the 5 '-terminal 13 S fragment [24,25].…”

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

“…Under defined conditions of mild nuclease digestion, 23S RNA within intact 50S ribosomal subunits can be cleaved into three fragments of -1200, 800 and 1000 nucleotides derived, respectively, from the 5' terminal, central and 3' terminal portions of the native molecule (Allet and Spahr, 1971;Spierer et al, 1975Spierer et al, , 1976. By hindsight, these RNA fragments appear to encompass the folded domains I plus II, Ill plus IV and V plus VI respectively.…”

“…We therefore decided to undertake such studies in the belief that protein L2 might lead us to another portion of 23S rRNA, other than domain V, that is involved in the peptidyl transferase centre. We were also aware that protein L23 (the target for photo-incorporation of puromycin) had also been shown to bind, independently of L2, to that same central fragment of 23S RNA (Spierer et al, 1976). Accordingly, these two ribosomal proteins were used to protect specific portions of 23S RNA from nuclease digestion.…”

Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli

“…By screening of a genomic phage library from T. borreli, using the ATPase domain of the topoisomerase II (topoII) gene as a probe, we identified a 1.4‐kb DNA fragment containing the gene for ribosomal protein L3 (L3). L3 is vital for the function of the ribosome and has been shown to participate in or initiate the early steps of the ribosomal assembly [5], where it binds with high affinity to the 23S rRNA [6]. L3 is involved in the formation of the peptidyltransferase center and is essential for its catalytic activity [7–10].…”