Abstract
Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m2G966 and m5C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.
Highlights
RNA-protein and RNA-RNA interactions are necessary for the main cellular activities of every living organism [1]
The distribution of modified nucleotides appears to be random throughout the ribosomal RNA (rRNA) secondary structure, three-dimensional structures show that they are clustered in the functional center of the ribosome such as the peptidyl-transferase center (PTC), decoding region, aminoacyl- and peptidyl-tRNA binding sites (A and P sites), peptide exit tunnel, and intersubunit bridges [6,7]
We identified peptide ligands that bind to wild-type h31 and unmodified h31 by M13 bacteriophage display
Summary
RNA-protein and RNA-RNA interactions are necessary for the main cellular activities of every living organism [1]. Natural RNA modifications often play a role in modulating or fine-tuning these interactions [2]. Lack of modification N6,N6-dimethyladenosine at positions 1518 and 1519 of the small subunit ribosomal RNA (rRNA) alters the RNA structure and affects 30S subunit assembly [3,4], and tRNA modifications have been shown to fine-tune the affinity of the various tRNAs for their ribosome binding sites [5]. Resistance to antibiotics can arise if specific modifications are either lacking or inserted [13,14]. These facts highlight the possibility of using modified RNAs as potential drug targets. There is an urgent need for identification of new drug target sites because of the emergence of multiple drug resistance [15,16,17], as well as functional mutations at the existing target sites [18,19,20]
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