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 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 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.
Highlights
Most gene expression requires ribosome translation of mRNA sequences into polypeptides
The periodicity, measured by Log-Odds Ratios (LOR), was significantly elevated (Fig 2C) in the ramp Open Reading Frames (ORFs) regions of genes whose proteins were detected in cell lysates by peptide MS/MS spectrometry [32], suggesting that the periodicity is more pronounced in genes with high protein expression
This conclusion was confirmed by examining the ramp ORF periodicities of genes known to have high, intermediate and low protein expression (Fig 2D), based on expression levels reported in genome-wide Western analysis of 3,838 epitope-tagged yeast genes [33]
Summary
Most gene expression requires ribosome translation of mRNA sequences into polypeptides. Many features of this process are very well described, there are mechanisms governing translation rate, at the genomic scale, that remain to be discovered. Different mRNAs can vary 10–100 fold in their translation efficiency [1,2,3]. Translation efficiency is influenced by biophysical parameters and informational properties of the mRNA molecule. Low mRNA stability and strong secondary structure can have negative effects on translation efficiency that are largely independent of the sequence contained within the mRNA [4, 5]. Sequence determinants of translation efficiency are less well-described but are increasingly accepted as playing important roles in the translation mechanism in disparate taxa [6,7,8,9]
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