Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 3577-3587 |
| Number of pages | 11 |
| Journal | Journal of Molecular Biology |
| Volume | 428 |
| Issue number | 18 |
| DOIs | |
| Publication status | Published - 2016 Sep 11 |
| Externally published | Yes |
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Keywords
- protein synthesis
- recycling phase not obligatory
- ribosome recycling
- RRF function
- termination of protein synthesis
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology
Cite this
The Termination Phase in Protein Synthesis is not Obligatorily Followed by the RRF/EF-G-Dependent Recycling Phase. / Qin, Bo; Yamamoto, Hiroshi; Ueda, Takuya; Varshney, Umesh; Nierhaus, Knud H.
In: Journal of Molecular Biology, Vol. 428, No. 18, 11.09.2016, p. 3577-3587.Research output: Contribution to journal › Article
}
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.
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 - protein synthesis
KW - recycling phase not obligatory
KW - ribosome recycling
KW - RRF function
KW - termination of protein synthesis
UR - http://www.scopus.com/inward/record.url?scp=84984605008&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84984605008&partnerID=8YFLogxK
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 -