No abstract

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5ʹ UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3 rd to the 5 th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.

Translation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5' UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occurs. For some genes, translation elongation also affects the protein synthesis rate. Recently, it was proposed that the identity of codons three to five, called short translational ramp, have a strong influence on translation elongation and protein expression. By the use of a GFP library where nearly all combinations of nucleotides at these positions were created, it was demonstrated that some of nucleotides combinations increased GFP expression up to four orders of magnitude by enhancing their translation efficiency (TE). While it is clear that the short ramp can influence protein expression levels of artificial constructs, its impact on physiological proteins is still unknown. In this work, we aimed to investigate the relevance of the short translational ramp on a physiological context. Through bioinformatics analysis, we identified the nucleotide combinations from the GFP library on Escherichia coli genes and examined their correlation with TE. We observed that E. coli genes were enriched with nucleotide compositions that enhanced protein expression on the GFP library, but, surprisingly, it seems to affect the TE only marginally.Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of the short translational ramp.

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

Highly positively charged protein segments are known to result in poor translation efficiency by the action of the ribosome quality control complex (RQC). This effect may be explained by ribosome stalling caused by electrostatic interactions between the nascent peptide and the negatively charged ribosome exit tunnel. This leads to translation termination followed by activation of mRNA decay pathways and protein degradation by RQC. Polybasic peptides are mainly studied with reporter systems, where artificial sequences are introduced into heterologous genes. The unique example of an endogenous protein targeted by RQC is Rqc1, a protein essential for the RQC activity. RQC arguably regulates Rqc1 levels through a conserved polybasic domain present in its N-term. We aimed to check if RQC act as a regulatory mechanism for other endogenous proteins containing polybasic domains. Here we show by bioinformatics, ribosome profiling data, and western blot protein quantification that endogenous proteins containing polybasic domains similar to or even more positively charged than Rqc1 are not targeted by RQC, suggesting that endogenous polybasic domains are not sufficient to induce degradation by this complex. We further demonstrate that Rqc1 levels are not regulated by the RQC complex, but by Ltn1 alone, in a post-translational fashion.

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase, Rad6. How Rad6-mediated ubiquitination of ribosomes affects global translation, however, is unclear. We therefore performed Ribo-seq and Disome-seq in Saccharomyces cerevisiae, and found that oxidative stress caused ribosome pausing at specific amino acid motifs, and this also led to ribosome collisions. However, these redox pausing signatures were lost in the absence of Rad6 but did not depend on the ribosome-associated quality control (RQC) pathway. We also found that Rad6 is needed to inhibit overall translation in response to oxidative stress and its deletion leads to increased expression of antioxidant genes. Finally, we observed that the lack of Rad6 leads to changes during translation initiation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation.

No abstract

Protein synthesis is essential to support homeostasis, and thus, must be highly regulated during cellular response to harmful environments. All stages of translation are susceptible to regulation under stress, however, the mechanisms involved in translation regulation beyond initiation have only begun to be elucidated. Methodological advances enabled critical discoveries on the control of translation elongation, highlighting its important role in translation repression and the synthesis of stress-response proteins. In this article, we discuss recent findings on mechanisms of elongation control mediated by ribosome pausing and collisions and the availability of tRNAs and elongation factors. We also discuss how elongation intersects with distinct modes of translation control, further supporting cellular viability and gene expression reprogramming. Finally, we highlight how several of these pathways are reversibly regulated, emphasizing the dynamics of translation control during stress-response progression. A comprehensive understanding of translation regulation under stress will produce fundamental knowledge of protein dynamics while opening new avenues and strategies to overcome dysregulated protein production and cellular sensitivity to stress.