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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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Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.(A) Schematics of the mCherry (top) and luciferase (middle, and bottom) reporters used in this study. The mCherry reporter contains an N-terminal thioredoxin (Thrdx) domain, 3HA-tag, sequence of interest (black section), followed by the C-terminal mCherry sequence. The top luciferase reporter includes a 2HA tag followed by sequences of interest (used for study in Figure 1B). The second luciferase reporter (used in Figure 6) has sequences of interest at the N-terminal end of Renilla. Firefly is used in this construct as an internal control in the second luciferase reporter. (B) Relative amounts of protein expressed from reporters expressed in E. coli (mCherry, red) and S. cerevisiae (luciferase, green). Error bars results from for the standard error of at least three experiments.DOI:http://dx.doi.org/10.7554/eLife.05534.003
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fig1: Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.(A) Schematics of the mCherry (top) and luciferase (middle, and bottom) reporters used in this study. The mCherry reporter contains an N-terminal thioredoxin (Thrdx) domain, 3HA-tag, sequence of interest (black section), followed by the C-terminal mCherry sequence. The top luciferase reporter includes a 2HA tag followed by sequences of interest (used for study in Figure 1B). The second luciferase reporter (used in Figure 6) has sequences of interest at the N-terminal end of Renilla. Firefly is used in this construct as an internal control in the second luciferase reporter. (B) Relative amounts of protein expressed from reporters expressed in E. coli (mCherry, red) and S. cerevisiae (luciferase, green). Error bars results from for the standard error of at least three experiments.DOI:http://dx.doi.org/10.7554/eLife.05534.003

Mentions: To begin investigating the translation of poly(lysine)-encoding sequences, we created a series of mCherry- and luciferase-based reporter constructs (Figure 1A) containing no insert, glutamic acid (GAA) repeats, or consecutive lysine residues encoded by various combinations of AAA and AAG codons. These reporters were introduced into S. cerevisiae and E. coli cells and the protein products visualized by either luminescence or fluorescence, respectively (Figure 1B). The insertion of twelve consecutive negatively charged glutamic acid residues (GAA) had no negative impact on production of the reporter protein (Figure 1B). By contrast, the addition of consecutive lysine residues generally resulted in overall less protein production (Figure 1B), consistent with previous studies of poly(lysine)-containing reporters (Ito-Harashima et al., 2007; Lu and Deutsch, 2008; Chiabudini et al., 2012). Interestingly, we find that protein output from the poly(lysine)-containing reporters is codon dependent in both bacteria and yeast; reporters containing iterated AAG lysine codons generate more protein than those with an equivalent number of synonymous AAA codons (Figure 1B). The relative differences in expression of AAG- vs AAA-encoded poly(lysine)-containing reporters in E. coli and S. cerevisiae are comparable (4 ± 0.3-fold more in E. coli and 3 ± 1-fold more in S. cerevisiae from reporters with AAG12 vs AAA12).10.7554/eLife.05534.003Figure 1.Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.


Ribosomes slide on lysine-encoding homopolymeric A stretches.

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

Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.(A) Schematics of the mCherry (top) and luciferase (middle, and bottom) reporters used in this study. The mCherry reporter contains an N-terminal thioredoxin (Thrdx) domain, 3HA-tag, sequence of interest (black section), followed by the C-terminal mCherry sequence. The top luciferase reporter includes a 2HA tag followed by sequences of interest (used for study in Figure 1B). The second luciferase reporter (used in Figure 6) has sequences of interest at the N-terminal end of Renilla. Firefly is used in this construct as an internal control in the second luciferase reporter. (B) Relative amounts of protein expressed from reporters expressed in E. coli (mCherry, red) and S. cerevisiae (luciferase, green). Error bars results from for the standard error of at least three experiments.DOI:http://dx.doi.org/10.7554/eLife.05534.003
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.(A) Schematics of the mCherry (top) and luciferase (middle, and bottom) reporters used in this study. The mCherry reporter contains an N-terminal thioredoxin (Thrdx) domain, 3HA-tag, sequence of interest (black section), followed by the C-terminal mCherry sequence. The top luciferase reporter includes a 2HA tag followed by sequences of interest (used for study in Figure 1B). The second luciferase reporter (used in Figure 6) has sequences of interest at the N-terminal end of Renilla. Firefly is used in this construct as an internal control in the second luciferase reporter. (B) Relative amounts of protein expressed from reporters expressed in E. coli (mCherry, red) and S. cerevisiae (luciferase, green). Error bars results from for the standard error of at least three experiments.DOI:http://dx.doi.org/10.7554/eLife.05534.003
Mentions: To begin investigating the translation of poly(lysine)-encoding sequences, we created a series of mCherry- and luciferase-based reporter constructs (Figure 1A) containing no insert, glutamic acid (GAA) repeats, or consecutive lysine residues encoded by various combinations of AAA and AAG codons. These reporters were introduced into S. cerevisiae and E. coli cells and the protein products visualized by either luminescence or fluorescence, respectively (Figure 1B). The insertion of twelve consecutive negatively charged glutamic acid residues (GAA) had no negative impact on production of the reporter protein (Figure 1B). By contrast, the addition of consecutive lysine residues generally resulted in overall less protein production (Figure 1B), consistent with previous studies of poly(lysine)-containing reporters (Ito-Harashima et al., 2007; Lu and Deutsch, 2008; Chiabudini et al., 2012). Interestingly, we find that protein output from the poly(lysine)-containing reporters is codon dependent in both bacteria and yeast; reporters containing iterated AAG lysine codons generate more protein than those with an equivalent number of synonymous AAA codons (Figure 1B). The relative differences in expression of AAG- vs AAA-encoded poly(lysine)-containing reporters in E. coli and S. cerevisiae are comparable (4 ± 0.3-fold more in E. coli and 3 ± 1-fold more in S. cerevisiae from reporters with AAG12 vs AAA12).10.7554/eLife.05534.003Figure 1.Protein production is differentially diminished by iterated lysine codons (AAA vs AAG) in E. coli and S. cerevisiae.

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