Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Abstract

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex

“…We observed that polybasic sequences led to slower ribosome movement during translation and an increase in disome formation. However, we could not detect signatures of severe ribosome stalling, such as the presence of short (20-22 nt) mRNA fragments which indicates empty A site in ribosomes [39]. These data suggested that polybasic sequences of endogenous proteins do not lead to RQC complex activation.…”

“…Recently, it was shown that ribosomes with open A sites produce shorter mRNA fragments (20-22 nucleotides) than ribosomes with occupied A sites, which produce the longer mRNA fragments (27)(28)(29) nucleotides) analyzed in Figure 3a [35]. Structural and biochemical evidence demonstrated that the electrostatic repulsion caused by the presence of a polybasic sequence inside the exit tunnel can decrease the binding efficiency of an incoming tRNA carrying an additional positively charged residue, leading to empty A site ribosome [25,39,68]. Since ribosomes with empty A-site are recognized by translation termination factors, the analysis of 20-22 fragments could more accurately reflect translation efficiency [39].…”

“…Structural and biochemical evidence demonstrated that the electrostatic repulsion caused by the presence of a polybasic sequence inside the exit tunnel can decrease the binding efficiency of an incoming tRNA carrying an additional positively charged residue, leading to empty A site ribosome [25,39,68]. Since ribosomes with empty A-site are recognized by translation termination factors, the analysis of 20-22 fragments could more accurately reflect translation efficiency [39]. The ICP was the only group that showed an accumulation of short reads associated to empty A sites (Figure 3b).…”

Improved computational analysis of ribosome dynamics from 5’P degradome data using fivepeseq

“…To ease comparison of translational features between samples, we also generate differential heatmaps for each sample pair, where the normalized difference between counts at every position from each codon is displayed ( Figure 3B,C). As certain combinations of codons or amino acids can also affect ribosome dynamics (35,36), we additionally analyzed the ribosome protection associated with the presence of two and three codon combinations (and di/tripeptide) ( Figure 3D-E).…”

“…In addition to single amino acids, specific arrangement of consecutive amino acids can also lead to ribosome stalls, as the neighboring amino acids affect peptide bond formation or modulate interactions in the ribosome exit tunnel (35). In other cases, specific codon motifs can change the mRNA conformation, interfering with the decoding process (36). To facilitate the exploration of this phenomenon, we generate an automatic report investigating two or three codon (or amino acids) combinations able to produce ribosome stalls.…”