GCN sensitive protein translation in yeast

Abstract

MPW) ¶ These authors contributed equally to this work. AbstractLevels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN) n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with largescale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

“…Alternatively, increasing H-bonding to the CAR surface could make ribosome-mRNA interaction too ‘sticky’, possibly hindering the threading of the mRNA and reducing translocation efficiency. We hypothesize that both scenarios are true, depending on the strength of the mRNA–CAR interactions and translation level of the gene [ 8 ]. For low-expression genes, which tend to have low or modest GCN periodicity in their ramp, increasing mRNA–CAR interactions may promote translation, whereas for high-expression genes, which tend to have strong GCN periodicity, increased mRNA–CAR interactions may depress translation.…”

“…For low-expression genes, which tend to have low or modest GCN periodicity in their ramp, increasing mRNA–CAR interactions may promote translation, whereas for high-expression genes, which tend to have strong GCN periodicity, increased mRNA–CAR interactions may depress translation. In this model [ 8 ], increased mRNA–CAR interactions under stress conditions would result in up- or down-regulation of translation, depending on a gene’s expression level and the strength of its ramp GCN periodicity. Since genes with high protein expression tend to have strong GCN periodicity in their ramp regions, translation of these genes would be depressed under stress conditions.…”

“…We were particularly interested in how mRNA sequences might interact with the surface of the ribosome’s mRNA entrance or exit tunnels, because the codons immediately following the translation start codon, referred to as the ‘ramp’ region [ 6 , 7 ], have a tendency to have a three-nucleotide repeating pattern; GCN (where N represents A, C, G or U) [ 8 ]. A subtle GCN periodicity has been detected throughout ORFs [ 9 , 10 ], and we have found that the GCN periodicity is particularly pronounced in the ramp regions of genes exhibiting high protein expression [ 8 ]. These observations raise the hypothesis that mRNA codons might dynamically interact with the mRNA tunnel surface before or after passing through the decoding center A and P sites.…”

Codon Dependency Of Ribosome Car Function

“…Previous work in the Weir lab found that the ramp region, codons 3 to 7 of the open reading frame (ORF), of yeast genes has an overrepresentation of G at codon position 1 and C at position 2 coupled with an underrepresentation of G at position 2 (Figure I.6A) (Barr et al, 2020). This GCN periodicity is more pronounced in highly expressed proteins.…”

“…Mass spectrometry detected proteins showed a depression of G at position 2 of codons 4 to 7 in highly expressed proteins (Figure I.6B) (Lycette et al, 2016). Additionally, an analysis of the mRNA sequence of the ramp region, showed that the top 10% of genes had more pronounced GCN periodicity with a depression of G at position 2 (Figure I.6C) (Barr et al, 2020). Thus, GCN periodicity is most pronounced in the ramp regions of highly expressed genes.…”