Role of Ribosome Recycling Factor in Translational Coupling as a Ribosome Releasing Factor

The objective of this presentation is to study the in vivo actions of ribosome recycling factor (RRF) and compare them with those found in vitro . RRF is known to catalyze three separate reactions: release of tRNA and mRNA from the post‐termination complex (PoTC), and splitting of the ribosome of the PoTC. To study the role of RRF in translational coupling in vivo , we used E. coli harboring temperature sensitive (ts) RRF and assayed by following downstream reading of translationally coupled ORF. At the non‐permissive temperature, ribosomes remain on the termination codon of the junction sequence of coupled ORFs and translate downstream ORF without the Shine‐Dalgarno (SD) sequence. The readings were in all three frames due to thermal frameshift at the termination codon. Introduction of sequence complementary to the 3′‐terminal of 16S rRNA into the region surrounding the junction sequence increased downstream reading. When upstream ORF was short, translation of the downstream reading was abolished, suggesting that the ribosomes released by RRF are moving toward the SD sequence of the upstream ORF. The thermal frame shift at the stop codon was also stopped by the upstream SD sequence. Our data suggest that the ribosome‐bound mRNA may take a secondary structure around the junction sequence. For in vitro studies, we used mRNA that incorporated different radioactively labeled amino acids based on the frameshift at the junction sequence, UAAUG, of two translationally coupled ORFs. In the absence of RRF, the ribosome stayed on the mRNA and translated in frame with the termination codon UAA. In the presence of RRF, amino acid incorporation occurred in frame with the start codon AUG. This suggests that RRF releases the ribosome from UAA and the released ribosome binds to AUG and begins translation. With the use of tethered, unsplittable ribosomes (Ribo‐T) in the in vitro system, we showed that complete ribosomal splitting is not required for the action of RRF in translational coupling. Therefore, the main role of RRF in translational coupling appears to be the release of ribosomes from mRNA. Support or Funding Information Supported and funded by Creative Biomedical Research Institute (CBRI) in Philadelphia, Pennsylvania. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .