Abstract
In bacteria, stop codons are recognized by two similar class 1 release factors, release factor 1 (RF1) and release factor 2 (RF2). Normally, during termination, the class 2 release factor 3 (RF3), a GTPase, functions downstream of peptide release where it accelerates the dissociation of RF1/RF2 prior to ribosome recycling. In addition to their canonical function in termination, both classes of release factor are also involved in a post peptidyl transfer quality control (post PT QC) mechanism where the termination factors recognize mismatched (i.e. error-containing) ribosome complexes and promote premature termination. Here, using a well defined in vitro system, we explored the role of release factors in canonical termination and post PT QC. As reported previously, during canonical termination, RF1 and RF2 recognize stop codons in a similar manner, and RF3 accelerates their rate of dissociation. During post PT QC, only RF2 (and not RF1) effectively binds to mismatched ribosome complexes; and whereas the addition of RF3 to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to RF1 inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation. Our data strongly suggest that RF2, in addition to its primary role in peptide release, functions as the principle factor for post PT QC.
Highlights
Bacteria have two similar release factors: release factor 1 and release factor 2 for terminating protein synthesis
During post PT QC, only release factor 2 (RF2) effectively binds to mismatched ribosome complexes; and whereas the addition of release factor 3 (RF3) to RF2 increased its rate of release on mismatched complexes, the addition of RF3 to release factor 1 (RF1) inhibited its rate of release but increased the rate of peptidyl-tRNA dissociation
RF1 and RF2 Act Differently on Mismatched Complexes—We first established that RF1 and RF2 behave during canonical termination in our in vitro system as reported previously [13]
Summary
Bacteria have two similar release factors: release factor 1 and release factor 2 for terminating protein synthesis. During post PT QC, a misincorporation event that results in a mismatched tRNA/mRNA interaction in the P site of the ribosome leads to substantial losses in fidelity for subsequent decoding steps These mismatched complexes are recognized by release factors and protein synthesis terminates prematurely. We show that whereas RF1 and RF2 display similar behaviors during canonical stop codon recognition, RF2 appears to be more efficient and broadly competent at promoting termination on mismatched complexes (post PT QC) These data suggest that RF2, in addition to its participation in canonical release, functions as a specialized quality control factor with RF3
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