TY - JOUR
T1 - The Termination Phase in Protein Synthesis is not Obligatorily Followed by the RRF/EF-G-Dependent Recycling Phase
AU - Qin, Bo
AU - Yamamoto, Hiroshi
AU - Ueda, Takuya
AU - Varshney, Umesh
AU - Nierhaus, Knud H.
N1 - Funding Information:
We thank Drs. Akira and Hideko Kaji for both supplying us with RRF antibodies and intensive discussions, and Drs. Markus Pech, Gene Center, University of Munich, Germany, and John Achenbach, NOXXON Pharma AG, Berlin, Germany, for help and discussions. We thank Profs. M. Vingron, Max-Planck-Institut für Molekulare Genetik in Berlin-Dahlem, and C.M.T. Spahn, Institute of Medical Physics and Biophysics at the Charité, Berlin, for generous support. This work was supported by the China Scholarship Council (File No. 20114911234 ) to B.Q., the HFSPO Research Grant Funds (HFSP-Ref. RGP0008/2014 ) to K.H.N. and T.U., and the Alexander-von-Humboldt Foundation (grant GAN 1127366 STP-2 ) to H.Y., the Max-Planck-Gesellschaft and the Verein zur Förderung junger Wissenschaftler (Berlin) .
Funding Information:
Financial Disclosure: This work was supported by the Max-Planck-Gesellschaft and the Verein zur Förderung junger Wissenschaftler (Berlin). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2016 Elsevier Ltd
PY - 2016/9/11
Y1 - 2016/9/11
N2 - It is general wisdom that termination of bacterial protein synthesis is obligatorily followed by recycling governed by the factors ribosomal recycling factor (RRF), EF-G, and IF3, where the ribosome dissociates into its subunits. In contrast, a recently described 70S-scanning mode of initiation holds that after termination, scanning of 70S can be triggered by fMet-tRNA to the initiation site of a downstream cistron. Here, we analyze the apparent conflict. We constructed a bicistronic mRNA coding for luciferases and showed with a highly resolved in vitro system that the expression of the second cistron did not at all depend on the presence of active RRF. An in vivo analysis cannot be performed in a straightforward way, since RRF is essential for viability and therefore, the RRF gene cannot be knocked out. However, we found an experimental window, where the RRF amount could be reduced to below 2.5%, and in this situation, the expression of the second cistron of a bicistronic luciferase mRNA was only moderately reduced. Both in vitro and in vivo results suggested that RRF-dependent recycling is not an obligatory step after termination, in agreement with the previous findings concerning 70S-scanning initiation. In this view, recycling after termination is a special case of the general RRF function, which happens whenever fMet-tRNA is not available for triggering 70S scanning.
AB - It is general wisdom that termination of bacterial protein synthesis is obligatorily followed by recycling governed by the factors ribosomal recycling factor (RRF), EF-G, and IF3, where the ribosome dissociates into its subunits. In contrast, a recently described 70S-scanning mode of initiation holds that after termination, scanning of 70S can be triggered by fMet-tRNA to the initiation site of a downstream cistron. Here, we analyze the apparent conflict. We constructed a bicistronic mRNA coding for luciferases and showed with a highly resolved in vitro system that the expression of the second cistron did not at all depend on the presence of active RRF. An in vivo analysis cannot be performed in a straightforward way, since RRF is essential for viability and therefore, the RRF gene cannot be knocked out. However, we found an experimental window, where the RRF amount could be reduced to below 2.5%, and in this situation, the expression of the second cistron of a bicistronic luciferase mRNA was only moderately reduced. Both in vitro and in vivo results suggested that RRF-dependent recycling is not an obligatory step after termination, in agreement with the previous findings concerning 70S-scanning initiation. In this view, recycling after termination is a special case of the general RRF function, which happens whenever fMet-tRNA is not available for triggering 70S scanning.
KW - RRF function
KW - protein synthesis
KW - recycling phase not obligatory
KW - ribosome recycling
KW - termination of protein synthesis
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U2 - 10.1016/j.jmb.2016.05.019
DO - 10.1016/j.jmb.2016.05.019
M3 - Article
C2 - 27261258
AN - SCOPUS:84984605008
VL - 428
SP - 3577
EP - 3587
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 18
ER -