Ribosome Pausing: Mechanisms and Implications in Translation
The process of translation is characterized by variation in the local decoding rates of specific mRNA codons. There are known cases where pausing or stalling is used for regulating protein synthesis. These are frequently found in the translated ORFs in 5’UTRs whereby the paused ribosome alters the progression of ribosomes to the CDS. Ribosome pausing has also been implicated in the cotranslational folding of proteins and for directing translation of peptides to endoplasmic reticulum. Below is a discussion of four papers that exemplify the impact that translatomic tools such as ribosome profiling can make in the area of ribosome pausing.
Selective inhibition of human translation termination by a drug-like compound
Nature communications, 2020, 11(1), pp.1-9.
Li, W., Chang, S.T.L., Ward, F. and Cate, J.H.
PF846 selectively inhibits the synthesis of specific proteins by stalling translation elongation. This strategy enables a potential expansion of the druggable proteome. Using cryo-EM, the authors investigated the mechanism of action of small molecules targeting translation.
- The authors were able to show that PF846 was capable of translation termination inhibition by introducing variants of randomized amino acids near the peptidyl transferase centre. This revealed that PF846 prevented the nascent chain from being released from the ribosome when in the presence of a Asn-Pro-Asn motif proceeding the stop codon. The authors isolated complexes containing this Asn-Pro-Asn motic and used cryo-EM to determine the conformation of PF846 stalled complexes.
- In PF846-stalled termination complexes, the nascent chain adopts an alpha helix formation prior to the Asn-Pro-Asn motif. This induces 28S rRNA nucleotide rearrangements that suppress the peptidyl transferase centre (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1).
PF846 induces stalling of translation termination giving rise to the possibility of small molecules – such as PF846 – to be used as selective inhibitiors of disease-causing products by stalling their translation. The ability to inhibit protein synthesis in a highly selective manner dependent on the nascent polypeptide sequence opens new ways to target proteins. The paper provides a good insight into how sequence specificity of drug-like compounds can be used to aid the development of these small molecules for therapeutic interest.
Molecular mechanism of drug-dependent ribosome stalling
Molecular cell, 2008, 30(2), pp.190-202.
Vazquez-Laslop, N., Thum, C. and Mankin, A.S.
During translation, the nascent peptide is threaded through the exit tunnel. Occasionally the nascent peptide interacts with this conduit, which is predominantly rRNA. These interactions can induce stalling within the ribosome. In this paper, the authors investigated the stalling of the ribosome during translation of the peptide ErmC and its reliance on macrolide antibiotic-dependent ribosome stalling. The authors used a cell-free transcription-translation system to look into the structure of stalled complexes within the exit tunnel.
- The nascent peptide sequence that is responsible for pauses consists of a 9 amino acid-long peptide MGIFSIFVI with the isoleucine positioned in the P/P site of the arrested ribosome, i.e. ribosome stalls after the translocation step.
- The nascent peptide forms a structure that prevents catalysis of peptide bond formation.
- The authors show a C3 cladinose sugar is required by the macrolide antibiotic to induce stalling. This sugar causes the exit tunnel of the ribosome to narrow, which positions the nascent peptide for interaction with the tunnel sensory elements
The picture that emerges from comparison of ErmCL with other known stalling peptides (SecM, TnaC) is that the key components of the stalling mechanism are similar and involve specific elements of the ribosome tunnel, the nascent peptide, and the PTC. In all three systems the activity of the PTC is inhibited and the identity of the amino acid residues present in the P site of the PTC is critical for stalling
eIF5A functions globally in translation elongation and termination
Molecular cell, 2017, 66(2), pp.194-205.
Schuller, A.P., Wu, C.C.C., Dever, T.E., Buskirk, A.R. and Green, R.
eIF5A (eukaryotic translation initiation factor 5A) is an mRNA-binding protein that is involved in translation elongation and has an important role at the level of mRNA turnover. It is a small, highly expressed protein containing only 157 amino acids. eIF5A is post translationally modified with hypusine at a conserved lysine residue. This modification is critical for eIF5A function as hypusine is required for eIF5A to associate with ribosomes. The authors utilised ribosome profiling to investigate an eIF5A-depleted yeast strain.
- Depletion of eIF5A leads to global translation elongation defects.
- By inhibiting transcription of eIF5A, there was a large increase in the fraction of ribosomes close to the start codon relative to a wild type strain.
- This accumulation of ribosomes is consistent with the pauses in elongation followed by queuing of upstream ribosomes.
- eIF5A promotes translation termination.
- The stop codon peak in the eIF5A depleted strain is approx. 2-fold higher than the wild type strain and is also accompanied by a secondary peak approx. 30 nucleotides upstream.
- Ribosomes were seen to accumulate at stop codons and in the 3’ UTR, suggesting a global defect in termination when eIF5A is absent/depleted.
The authors show that eIF5A is a strong promoter of translation in cases of stalled ribosomes, with a global role in alleviating widespread elongation pausing. This global role would explain its essential nature in eukaryotic cells and its abundance within those cells, with it being in the top 50 highest expressed genes in S. cerevisiae, S. pombe and HeLa cells.
Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing
Molecular cell, 2018, 70(2), pp.254-264.
Ivanov, I.P., Shin, B.S., Loughran, G., Tzani, I., Young-Baird, S.K., Cao, C., Atkins, J.F. and Dever, T.E.
Polyamines are small organic polycations that are essential for a variety of basic cellular functions. The first stage of polyamine synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). Antizyme inhibitor (AZIN) is a homologue for ODC that regulates polyamine synthesis. The translation of AZIN is inversely correlated with translation of aN upstream ORF that has a AUU start codon. The authors of this report use ribosome profiling to reveal the mechanism underlying polyamine regulation of AZIN synthesis.
- The coding sequence of AZIN1 is required for polyamine stimulation of ORF translation initiation.
- The presence of a pause in AZIN1 ORF enhances recognition of the ORF start codon AUU.
- Pausing at AZIN1 ORF depends on polyamines influence on eIF5A. The influence may be direct (eIF5A and polyamines compete for a common site on the ribosome) or by some other mechanism.
- Ribosome queuing upstream of paused elongating ribosomes can enhance selection of weak start codons.
This study shows that a paused elongating ribosome promotes translation initiation at an upstream near-cognate start codon. The mechanism is employed for regulation of AZIN1, but may be also used for regulation of other near cognate start codons.
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