Abstract
The succession of molecular events leading to eukaryotic translation reinitiation—whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA—is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR–MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR–MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.
Highlights
Eukaryotic translation reinitiation is a process that is only recently becoming understood at the mechanistic and functional levels
In some cases, when there is a short upstream open reading frame that precedes the main ORF, ribosomes can translate the uORF, terminate translation, and undergo a poorly understood process called “translation reinitiation” whereby they resume scanning for another AUG initiation codon and translate the main ORF
We previously showed that two noncanonical initiation factors, density-regulated reinitiation and release factor (DENR) and multiple copies in T
Summary
Eukaryotic translation reinitiation is a process that is only recently becoming understood at the mechanistic and functional levels. After recruitment of the ribosomal 40S subunit to the mRNA, often via the 50 cap, the 40S scans to locate the first appropriate AUG start codon for joining of the 60S subunit and commencement of translation. After translating this open reading frame (ORF) and terminating, the 60S subunit dissociates from the mRNA, leaving the 40S subunit bound to the mRNA. When the translated ORF is short, as is the case for many upstream open reading frames (uORFs), the 40S can remain bound to the mRNA, resume scanning, and reinitiate translation at a downstream ORF [1]. Recent ribosome profiling studies have found pervasive translation of uORFs [2,3,4], indicating that in many of these cases, translation reinitiation is important for permitting translation of the main ORF on the mRNA
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