The Termination Phase in Protein Synthesis is not Obligatorily Followed by the RRF/EF-G-Dependent Recycling Phase.
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.
Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany; Institut für Medizinische Physik und Biophysik, Charité, Charitéplatz 1, 10117 Berlin, Germany.
Bo
B
Qin
Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany; Institut für Medizinische Physik und Biophysik, Charité, Charitéplatz 1, 10117 Berlin, Germany. Electronic address: yamamoto.hiroshi@charite.de.
Hiroshi
H
Yamamoto
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba Prefecture 277-8562, Japan.
Takuya
T
Ueda
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Umesh
U
Varshney
Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany; Institut für Medizinische Physik und Biophysik, Charité, Charitéplatz 1, 10117 Berlin, Germany.
Knud H
KH
Nierhaus
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005796:Genes; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008954:Models, Biological; D010443:Peptide Chain Termination, Translational; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D020653:Peptide Elongation Factor G; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; D008156:Luciferases
RRF function; protein synthesis; recycling phase not obligatory; ribosome recycling; termination of protein synthesis