1P557 Peptide bond formation induces the breakage of Shine-Dalgarno interaction on the ribosome(26. Single molecule biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

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1P543 Single-molecule structural and functional analyses of nuclear pore complex(26. Single molecule biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Seibutsu Butsuri ◽

10.2142/biophys.46.s282_3 ◽

2006 ◽

Vol 46 (supplement2) ◽

pp. S282

Author(s):

Shotaro Otsuka ◽

Hirohide Takahashi ◽

Shige H. Yoshimura

Keyword(s):

Single Molecule ◽

Nuclear Pore Complex ◽

Poster Session ◽

Nuclear Pore ◽

Single Molecule Biophysics ◽

Meeting Program ◽

Functional Analyses

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1P534 Loading direction controls the ADP affinity of myosin V.(26. Single molecule biophysics,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Seibutsu Butsuri ◽

10.2142/biophys.46.s280_2 ◽

2006 ◽

Vol 46 (supplement2) ◽

pp. S280

Author(s):

Yusuke Oguchi ◽

Sergey V. Mikhailenko ◽

Adrian O. Olivares ◽

Enrique M. De La Cruz ◽

Shin'ichi Ishiwata

Keyword(s):

Single Molecule ◽

Poster Session ◽

Myosin V ◽

Single Molecule Biophysics ◽

Meeting Program ◽

Loading Direction

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Elongation Factor P Interactions with the Ribosome Are Independent of Pausing

mBio ◽

10.1128/mbio.01056-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 2

Author(s):

Rodney Tollerson ◽

Anne Witzky ◽

Michael Ibba

Keyword(s):

Single Molecule ◽

Bond Formation ◽

Elongation Factor ◽

Peptide Bond ◽

Cellular Distribution ◽

Peptide Bond Formation ◽

Translation Elongation ◽

Single Molecule Tracking ◽

Localization Pattern ◽

Elongation Factor P

ABSTRACT Bacterial elongation factor P (EF-P) plays a pivotal role in the translation of polyproline motifs. To stimulate peptide bond formation, EF-P must enter the ribosome via an empty E-site. Using fluorescence-based single-molecule tracking, Mohapatra et al. (S. Mohapatra, H. Choi, X. Ge, S. Sanyal, and J. C. Weisshaar, mBio 8:e00300-17, 2017, https://doi.org/10.1128/mBio.00300-17 !) monitored the cellular distribution of EF-P and quantified the frequency of association between EF-P and the ribosome under various conditions. Findings from the study showed that EF-P has a localization pattern that is strikingly similar to that of ribosomes. Intriguingly, EF-P was seen to bind ribosomes more frequently than the estimated number of pausing events, indicating that E-site vacancies occur even when ribosomes are not paused. The study provides new insights into the mechanism of EF-P-dependent peptide bond formation and the intricacies of translation elongation.

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