Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.
The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.
Noah
James W
JW
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Shapkina
Tatjana G
TG
Nanda
Kavita
K
Huggins
Wayne
W
Wollenzien
Paul
P
D000107:Acetylation / Q000528:radiation effects; D001665:Binding Sites / Q000528:radiation effects; D003596:Cytosine / Q000737:chemistry / Q000528:radiation effects; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D010441:Peptide Chain Elongation, Translational / Q000235:genetics / Q000528:radiation effects; D010777:Photochemistry; D011072:Poly U / Q000737:chemistry / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D012336:RNA, Ribosomal, 16S / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D014158:Transcription, Genetic / Q000528:radiation effects; D014466:Ultraviolet Rays
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012360:RNA, Transfer, Phe; D011072:Poly U; D003596:Cytosine