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Ribosomes slide on lysine-encoding homopolymeric A stretches.

Koutmou KS, Schuller AP, Brunelle JL, Radhakrishnan A, Djuranovic S, Green R - Elife (2015)

Bottom Line: 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.Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, United States.

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.

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Related in: MedlinePlus

Model for events during ribosome sliding.In this model translation is paused following the addition of the first lysine. The ribosome can than either slide or perform another round of peptide bond formation. If an AAA codon is positioned in the A site after sliding, the next step will also be slow, while if sliding results in a non-lysine codon in the A site, recovery from slow elongation may occur.DOI:http://dx.doi.org/10.7554/eLife.05534.020
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fig7: Model for events during ribosome sliding.In this model translation is paused following the addition of the first lysine. The ribosome can than either slide or perform another round of peptide bond formation. If an AAA codon is positioned in the A site after sliding, the next step will also be slow, while if sliding results in a non-lysine codon in the A site, recovery from slow elongation may occur.DOI:http://dx.doi.org/10.7554/eLife.05534.020

Mentions: Our biochemical data in E. coli lead us to propose a model (Figure 7) for what happens to the ribosome during the translation of homopolymeric A sequences. On these messages, the first lysine is added quickly (k1,obs) while subsequent lysines are added more slowly, causing the ribosome to pause. We note that the rate constants measured in the in vitro assay reflect all of the processes that can occur each time a new lysine moiety is added to the growing polypeptide chain (Lys-tRNALys binding, peptidyl-transfer, translocation, peptidyl-tRNA drop-off, 70S complex instability, etc). We suspect it to be unlikely that ribosome pausing is caused solely by dramatically large defects in peptidyl-transfer, but instead may result from ribosomes that become effectively inactivated (e.g. as a result of complex instability on homopolymeric A messages, etc). Whatever the cause for an initial ribosome pausing event on iterated AAA sequences, the ribosome can either slide or perform another round of peptide bond formation. If the ribosome slides such that another AAA codon is positioned in the A site, the next step will also be slow, while if sliding somehow positions a non-lysine codon in the A site, recovery from slow elongation may occur. In our in vitro system translating di-lysine messages, we are able to observe sliding when consecutive AAA-codons are present because we force a strong pause after MKK formation by leaving out downstream factors required for translation to proceed (Figure 3). Our data suggest that ribosome sliding on iterated AAA sequences is the major difference between the translation of poly(AAA)- and poly(AAG)-containing messages that results in substantially different protein outputs. While each sequential addition of lysine in an iterated AAG sequence may be slow, the ribosome maintains frame and ultimately is able to produce full-length protein. By contrast, with repeated AAA sequences, the ribosome can eventually escape the homopolymeric A sequence through repeated sliding events, often emerging out-of-frame from the A stretch, and thus unable to produce full-length protein.10.7554/eLife.05534.020Figure 7.Model for events during ribosome sliding.


Ribosomes slide on lysine-encoding homopolymeric A stretches.

Koutmou KS, Schuller AP, Brunelle JL, Radhakrishnan A, Djuranovic S, Green R - Elife (2015)

Model for events during ribosome sliding.In this model translation is paused following the addition of the first lysine. The ribosome can than either slide or perform another round of peptide bond formation. If an AAA codon is positioned in the A site after sliding, the next step will also be slow, while if sliding results in a non-lysine codon in the A site, recovery from slow elongation may occur.DOI:http://dx.doi.org/10.7554/eLife.05534.020
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363877&req=5

fig7: Model for events during ribosome sliding.In this model translation is paused following the addition of the first lysine. The ribosome can than either slide or perform another round of peptide bond formation. If an AAA codon is positioned in the A site after sliding, the next step will also be slow, while if sliding results in a non-lysine codon in the A site, recovery from slow elongation may occur.DOI:http://dx.doi.org/10.7554/eLife.05534.020
Mentions: Our biochemical data in E. coli lead us to propose a model (Figure 7) for what happens to the ribosome during the translation of homopolymeric A sequences. On these messages, the first lysine is added quickly (k1,obs) while subsequent lysines are added more slowly, causing the ribosome to pause. We note that the rate constants measured in the in vitro assay reflect all of the processes that can occur each time a new lysine moiety is added to the growing polypeptide chain (Lys-tRNALys binding, peptidyl-transfer, translocation, peptidyl-tRNA drop-off, 70S complex instability, etc). We suspect it to be unlikely that ribosome pausing is caused solely by dramatically large defects in peptidyl-transfer, but instead may result from ribosomes that become effectively inactivated (e.g. as a result of complex instability on homopolymeric A messages, etc). Whatever the cause for an initial ribosome pausing event on iterated AAA sequences, the ribosome can either slide or perform another round of peptide bond formation. If the ribosome slides such that another AAA codon is positioned in the A site, the next step will also be slow, while if sliding somehow positions a non-lysine codon in the A site, recovery from slow elongation may occur. In our in vitro system translating di-lysine messages, we are able to observe sliding when consecutive AAA-codons are present because we force a strong pause after MKK formation by leaving out downstream factors required for translation to proceed (Figure 3). Our data suggest that ribosome sliding on iterated AAA sequences is the major difference between the translation of poly(AAA)- and poly(AAG)-containing messages that results in substantially different protein outputs. While each sequential addition of lysine in an iterated AAG sequence may be slow, the ribosome maintains frame and ultimately is able to produce full-length protein. By contrast, with repeated AAA sequences, the ribosome can eventually escape the homopolymeric A sequence through repeated sliding events, often emerging out-of-frame from the A stretch, and thus unable to produce full-length protein.10.7554/eLife.05534.020Figure 7.Model for events during ribosome sliding.

Bottom Line: 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.Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, United States.

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.

Show MeSH
Related in: MedlinePlus