Identification of 50S Components Neighboring 23S rRNA Nucleotides A2448 and U2604 within the Peptidyl Transferase Center of Escherichia coli Ribosomes

Abstract

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

“…A second problem is the possibility of additional binding to secondary lower-affinity binding sites when excesses of oligonucleotide are used over 30 S subunits ( Figure 2B). Similar non-plateau binding curves have been reported in a previous study involving the use of excess photolabelled OMe oligonucleotides binding and cross-linking to the peptidyl transferase centre of 50 S subunits [18]. Thus filter binding appears to be inadequate for measurement of dissociation constants over a wide affinity range.…”

“…Filter binding has been the primary method used to date for assessing the binding of oligonucleotides to 50 S ribosomal subunits [18,19]. We found that where excess oligonucleotide was used (Figure 2B), some of the 10-mer oligonucleotides did not reach a plateau in the graph where the level of filter retention was titrated against a fixed concentration of 30 S subunits, particularly for weaker interactions.…”

“…This technique has been applied to binding of photolabile OMe oligonucleotides to 50 S ribosomal subunits [18,19], but not previously to 30 S subunits. We therefore assessed the binding strengths of the oligomers using the filterbinding technique.…”

“…However, the binding constants of oligodeoxyribonucleotides to RNA are poor, they are unstable to cellular nucleases and also form substrates for RNase H upon binding to the rRNA. A number of ribosome photochemical cross-linking studies have been carried out using oligoribonucleotides [16,17] as mRNA analogues or their OMe (2 -O-methyl) analogues [18] derivatized with photolabile groups as antisense agents. Such reagents were found to be more effective as ribosome-targeting agents than previously used oligodeoxyribonucleotide derivatives [19] due to their higher binding to RNA.…”

NUCLEAR FUSION DEFECTIVE1 Encodes the Arabidopsis RPL21M Protein and Is Required for Karyogamy during Female Gametophyte Development and Fertilization

“…In E. coli, L21 binds to 23S ribosomal RNA (Alexander and Cooperman, 1998;Vladimirov et al, 2000), which plays a direct role in protein synthesis (Samaha et al, 1995). Thus, nfd1 mutants are likely to be affected in protein synthesis within the mitochondrion.…”