Ribosomes slide on lysine-encoding homopolymeric A stretches

Abstract

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation.DOI: http://dx.doi.org/10.7554/eLife.05534.001

Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions

“…In our model, the re-pairing of the peptidyl-tRNA to the correct position on the mRNA during scanning may be stabilized by the SD-like sequence or a possible downstream 3’ stem-loop (Samatova et al, 2014); the SD-like sequence has a moderate effect on bypassing but may be important for the fidelity of landing site selection (Herr et al, 2004; Wills et al, 2008). All of these events happen during the rotated state pause; the majority of the pause is the ribosome sampling and exploring the reading frame widely, with movements possibly similar to the excursions and sliding behaviors observed previously (Koutmou et al, 2015; Yan et al, 2015). In the mechanism proposed here, bypassing is not induced by A-site (UAG stop codon) starvation, explaining why the absence of RF1 did not significantly affect the bypassing efficiency (Herr et al, 2000b).…”

Rapid generation of hypomorphic mutations

“…This method is based on polyA tracks, a novel cis regulatory element that decreases gene expression by disrupting messenger RNA (mRNA) translation1617. Insertion of consecutive adenosine nucleotides into the open reading frame of an mRNA will decrease protein expression by decreasing the efficiency of the translation elongation phase leading to diminished production of protein and mRNA destabilization, and thus to diminished mRNA levels.…”

Increased ribosome density associated to positively charged residues is evident in ribosome profiling experiments performed in the absence of translation inhibitors

“…Polybasic proteins cause similar effects, as demonstrated by our analysis and by different groups. [4][5][6][25][26][27] Our analysis, together with other recent studies exploring the effect of CHX on ribosome profiling, 23,28 showed, that the RP methodology can be more faithfully used to explore ribosomal translation if performed in the absence of translational inhibitors such as cycloheximide and anisomycin. We demonstrated that drug-free RP datasets tend to agree with the experimental evidence obtained by other methods analyzing the role of positively charged amino acids in reducing protein translation.…”

“…Ribosome sliding occurs on mRNAs regions that are rich in homopolymeric-A stretches and leads to mRNA degradation, frameshifts and alterations in protein output. 25,26 Since Homopolymeric-A stretches can be associated with translation of lysine residues, consequently ribosome sliding might be related to its charge. However, it is important to note that homopolymeric-A stretches do not necessarily code only for lysines.…”