eIF3g binding to GUCG boxes spread in mRNA coding regions mediates translational heat shock response
Heat shock response (HSR) is crucial for life's adaptation to heat. Here, we discovered a novel mechanism of HSR controlled at the translational level, which depends on the multi-subunit translation initiation factor, eIF3. The cis-element for this regulation is called GUCG box and occurs degenerately within protein-coding regions of regulated mRNAs. Being positioned at ribosome leading-edge through ?-propeller subunit eIF3i, the RNA-binding subunit eIF3g binds the motif and stabilizes mRNA anchoring during translation under mild heat. The eIF3g/i-regulated HSR consists of 64 genes including heat shock protein genes, engaging a quarter of the cellular ribosomes. The 5'-terminal coding regions of the HSR mRNAs are enriched with more GUCG boxes, some of which are masked by weak secondary structures.  This arrangement enables eIF3g/i-dependent enhancement in part through melting inhibitory secondary structures. GUCG boxes spread evenly across the entire protein-coding regions of strongly translated mRNAs ~20 nt. apart, as if to prevent ribosome collision. Thus, eIF3 not only stabilizes initiating ribosomes at its leading-edge but may contribute to anchoring elongating ribosomes throughout coding regions under heat insult. Overall design: SELEX was performed for 12 rounds in each experiment, as described  in 5, with 18 base-long randomized ribonucleotide sequence flanked by primer binding sites designed for cDNA synthesis, PCR and RNA synthesis by T7 RNA polymerase. For these experiments we used His6-tagged eIF3g/Tif35p or its complex with (untagged) eIF3i/Tif34p. The DNA fragments encoding them were amplified by PCR using budding yeast Saccharomyces cerevisiae genomic DNA and then cloned into pET28b (with hexa-histidine tag fused to its N-terminus, in the case of eIF3g) and were co-expressed in E. coli strain BL21-CodonPlus (DE3)-RILP (Stratagene).  The expressed protein(s) was purified by Ni-NTA (Qiagen) and Q-sepharose (Cytiva).