Binding and cross-linking of tmRNA to ribosomal protein S1, on and off the Escherichia coli ribosome.
UV irradiation of an in vitro translation mixture induced cross-linking of 4-thioU-substituted tmRNA to Escherichia coli ribosomes by forming covalent complexes with ribosomal protein S1 and 16S rRNA. In the absence of S1, tmRNA was unable to bind and label ribosomal components. Mobility assays on native gels demonstrated that protein S1 bound to tmRNA with an apparent binding constant of 1 x 10(8) M(-1). A mutant tmRNA, lacking the tag coding region and pseudoknots pk2, pk3 and pk4, did not compete with full-length tmRNA, indicating that this region is required for S1 binding. This was confirmed by identification of eight cross-linked nucleotides: U85, located before the resume codon of tmRNA; U105, in the mRNA portion of tmRNA; U172 in pK2; U198, U212, U230 and U240 in pk3; and U246, in the junction between pk3 and pk4. We concluded that ribosomal protein S1, in concert with the previously identified elongation factor EF-Tu and protein SmpB, plays an important role in tmRNA-mediated trans-translation by facilitating the binding of tmRNA to ribosomes and forming complexes with free tmRNA.
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Department of Animal and Dairy Sciences, Program in Cell and Molecular Biosciences, Auburn University, Auburn, AL 36849-5415, USA. iwower@acesag.auburn.edu
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Guven
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D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003062:Codon; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003062:Codon; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; C092408:smpB protein, Staphylococcus hominis; C096222:tmRNA; D010444:Peptide Elongation Factor Tu