In Vivo Studies of Function of RRF (Ribosome Recycling Factor) with Translationally Coupled Two ORFs Revealed Its Major Function: Release of mRNA from the Post‐Termination Complex

Abstract

ObjectiveStudy the RRF reaction in vivo to correct wrong prevalent concept that RRF's major function is to split ribosomes of post‐termination complexes.MethodsUse translationally coupled two ORFs and determine how much of the upstream ribosomes is reading the downstream ORF using the reporter gene. RRF is known to be essential for the translational coupling. We control RRF activity using tsRRF.ResultsIn the translationally coupled 2 ORFs, the downstream ORF is exclusively read by the ribosomes reading the upstream ORF. No free ribosomes participate in the reading of the downstream ORF. In this system, the border region of the two ORFs is such that termination codon of the upstream ORF and the initiation codon of the downstream ORF overlap as in UAAUG. At this junction, ribosomes just completed reading the upstream are released by RRF and about 25% of the released ribosomes re‐bind to the AUG and start translating the downstream gene. For this reason, the system offers unique opportunity to study the fate of ribosome finishing one round of translation which is under the control of ribosome recycling Factor (RRF). We placed this system in a strain (LJ14) which contains the temperature sensitive RRF so that one can study what happens in the absence and presence of RRF. Using this system, we showed 1) The major function of RRF is to release the ribosomes from mRNA but not splitting the ribosome. 2) When Ribosomes are not released due to the inactivation of RRF, ribosomes at UAA undergoes thermal frame shift and read all three frames. 3) For the ribosome released by RRF from UAA, AUG but not other triplet functions to read the downstream ORF. 4) This binding of released ribosomes occurs only if the AUG is present near UAA. 5) Introduction of short sequence hybridizable with ribosomal RNA helps ribosomes to rebind to the AUG. 6) When the upstream sequence is extremely short (4 codon or less), the ribosomes released from UAA by RRF rebinds to the upstream AUG with Shine Dalgarno (SD) sequence and the reading of the downstream ORF does not occur.ConclusionsOur results show that the early workers were led to a wrong concept that RRF does not release mRNA from the ribosome because they studied the action of RRF using short ORF with SD. Therefore, our original concept for which RRF was discovered as “ribosome releasing factor” was correct. The current wrong concept of the RRF action or ribosome recycling factor in general including the eukaryotes stems from the use of short ORF in in vitro studies in the early days.Support or Funding InformationFunded by Creative Bio‐medical Research Institute, Phildelphia Pa 19147