Antisense oligonucleotides targeting universally conserved 26S rRNA domains of plant ribosomes at different steps of polypeptide elongation.
A ribosome undergoes significant conformational changes during elongation of polypeptide chain that are correlated with structural changes of rRNAs. We tested nine different antisense oligodeoxynucleotides complementary to the selected, highly conserved sequences of Lupinus luteus 26S rRNA that are engaged in the interactions with tRNA molecules. The ribosomes were converted either to pre- or to posttranslocational states, with or without prehybridized oligonucleotides, using tRNA or mini-tRNA molecules. The activity of those ribosomes was tested via the so-called binding assay. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis. In this article, we present that (i) before and after translocation, fragments of domain V between helices H70/H71 and H74/H89 do not have to interact with nucleotides 72-76 of the acceptor arm of A-site tRNA; (ii) helix H69 does not have to interact with DHU arm of tRNA in positions 25 and 26 after forming the peptide bond, but before translocation; (iii) helices H69 and H70 interact weakly with nucleotides 11, 12, 25, and 26 of A-site tRNA before forming a peptide bond in the ribosome; (iv) interactions between helices H80, H93 and single-stranded region between helices H92 and H93 and CCAend of P-site tRNA are necessary at all steps of elongation cycle; and (v) before and after translocation, helix H89 does not have to interact with nucleotides in positions 64-65 and 50-53 of A-site tRNA TPsiC arm.
Bakowska-Zywicka
Kamilla
K
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland. bakowska@ibch.poznan.pl
Kietrys
Anna M
AM
Twardowski
Tomasz
T
D018390:Gene Targeting / Q000379:methods; D031297:Lupinus / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D010441:Peptide Chain Elongation, Translational / Q000235:genetics*; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry; D013329:Structure-Activity Relationship
D016376:Oligonucleotides, Antisense; D018749:RNA, Plant; D012335:RNA, Ribosomal; D012360:RNA, Transfer, Phe; C065096:RNA, ribosomal, 26S