Peptide bond formation destabilizes Shine-Dalgarno interaction on the ribosome.
The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence-a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNA(Phe), in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNA(Phe), in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNA(Phe), and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.
Uemura
Sotaro
S
Department of Physics, Stanford University, Stanford, California 94305, USA.
Dorywalska
Magdalena
M
Lee
Tae-Hee
TH
Kim
Harold D
HD
Puglisi
Joseph D
JD
Chu
Steven
S
D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics
D012333:RNA, Messenger; D012343:RNA, Transfer