GCN sensitive protein translation in yeast

Abstract

Levels 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 codonsthe 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 large-scale 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.

Arginine Methylation Regulates Ribosome CAR Function

“…R146 is conserved across eukaryotes, but not prokaryotes [ 12 , 18 ]. Mutational analysis has shown that when R146 is replaced in yeast Rps3, this can lead to lethal or slow growth phenotypes depending on the amino acid substitution, and R146 substitution can affect translation initiation and fidelity [ 39 , 40 ].…”

“…Since the CAR/mRNA interaction is highly sensitive to the sequence of the +1 codon, with a strong preference of GCN codons [ 12 , 18 ], the sensitivity of different mRNAs to regulation by CAR will depend upon its codon content. The initial codons of ORFs, the “ramp” region, appear to be particularly influential in modulating overall translation levels of mRNAs [ 41 ], perhaps analogous to an entry ramp to a highway determining traffic flow.…”

“…The initial codons of ORFs, the “ramp” region, appear to be particularly influential in modulating overall translation levels of mRNAs [ 41 ], perhaps analogous to an entry ramp to a highway determining traffic flow. Indeed, protein-coding ORFs tend to have a subtle three-nucleotide GCN periodicity [ 42 , 43 ], and many genes with high protein expression are particularly rich in GCN codons in their ramp regions suggesting that these genes have high sensitivity to CAR-mediated regulation [ 12 , 18 ].…”

“…Increasing the CAR/+1 codon interaction is thought to act like an accelerator or brake depending upon the protein expression levels of a gene and the degree of hydrogen bonding. Expression studies [ 18 ] suggest that increasing CAR/+1 interactions in the ramp regions of lower expression genes tends to increase their expression whereas the opposite is true for high-expression genes with GCN-rich codons in the ramp regions of their protein coding sequence. It is likely that future studies of CAR/mRNA interactions with different codons in the A-site and +1 positions, and the effects of ribosome modifications on these interactions, will provide further insights into this new layer of translation regulation.…”

“…Modulation of these interactions provides potential pathways for fine-tuning rates of protein translation in different cellular conditions. Cryo-EM studies in yeast have identified five intermediate stages of ribosome translocation (stages I through V; [ 17 ]) and we have identified a ribosome interaction surface, named CAR [ 12 , 18 ], that has pronounced hydrogen-bonding to the mRNA during the early translocation stages I and II. CAR interacts with the +1 codon next in line to enter the ribosome A site.…”