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Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation.

Pop C, Rouskin S, Ingolia NT, Han L, Phizicky EM, Weissman JS, Koller D - Mol. Syst. Biol. (2014)

Bottom Line: We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast.Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start.We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.

View Article: PubMed Central - PubMed

Affiliation: Computer Science Department, Stanford University, Stanford, CA, USA cpop@cs.stanford.edu.

No MeSH data available.


Comparison between codon translation rates in wild-type and mutantsCorrelation between estimated codon translation rates in wild-type versus mutant for the three mutant samples (the manipulated codon is highlighted in red). Rates are normalized by the minimum one in each sample. Pearson correlations are nearly exact, indicating that the mutant rates are generally unaffected.Source data are available online for this figure.
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fig03: Comparison between codon translation rates in wild-type and mutantsCorrelation between estimated codon translation rates in wild-type versus mutant for the three mutant samples (the manipulated codon is highlighted in red). Rates are normalized by the minimum one in each sample. Pearson correlations are nearly exact, indicating that the mutant rates are generally unaffected.Source data are available online for this figure.

Mentions: Our analysis of elongation rates on endogenous mRNAs in the context of the co-adapted cellular tRNA pool addresses the effects of codon usage in natural physiology, but may be confounded by this co-adaptation and cannot directly test the causal links between various correlated mRNA features. To measure the effect of tRNA abundance on codon translation rate directly, we created three mutant yeast species to test whether (i) tRNA overexpression speeds up translation, (ii) the tRNA body itself causes the tRNA-dependent rate effect observed in other studies, or (iii) depletion of tRNA slows down ribosomes. In our first mutant, AGG-OE, the tRNA recognizing AGG (namely tRNAArg(CCU)) was overexpressed on a high-copy plasmid; in mutant AGG-QC, the body sequence of the tRNA recognizing AGG was swapped with the body of a more preferred tRNA (as measured by tAI); and in mutant ACA-K, three out of four copies of the tRNA recognizing ACA were deleted from the genome. The AGG mutants had a URA marker and were compared against a wild-type sample with a URA plasmid (see Materials and Methods). For ACA-K, we checked that the abundance of the tRNA for ACA (namely tRNAThr(UGU)) did decrease to about 30% of wild-type (Supplementary Table S1). In the AGG-OE mutant, we measured the amount of total and aminoacylated tRNA for tRNAArg(CCU) (see Materials and Methods) and verified that the tRNA was overexpressed by 13.8-fold (±0.4), based on an analysis of two independently derived RNA samples, and remained charged at a level similar to wild-type (87%) (Supplementary Fig S2). For the AGG-QC mutant, we similarly verified that the amount of charged tRNAArg(CCU) was similar to wild-type (Supplementary Fig S2). We generated ribosome profiling data and ran our model on these mutants to test whether AGG codons are translated faster in AGG-OE and AGG-QC and whether ACA codons are translated slower in ACA-K. We observe no significant change in the elongation rates of the affected codon in any of the three mutants compared to wild-type (Fig3, Supplementary Fig S3); the overall correlation between ACA-K and wild-type is not as tight as for other mutants, but this is due to changes affecting all codons, not only ACA. We verified the result by inspecting the footprint counts at the perturbed codon relative to adjacent counts in the mutants compared to wild-type and saw no unusual increase or decrease (Supplementary Fig S4). One prevailing hypothesis (Welch et al, 2009) is that the amount of charged as opposed to total tRNA is the true predictor of codon elongation; our measurements of aminoacylated tRNA suggest that these levels were manipulated as expected and that this is not a confounding factor in the mutant samples. Hence, our results suggest that several-fold changes in tRNA abundance do not affect ribosome dwell time.


Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation.

Pop C, Rouskin S, Ingolia NT, Han L, Phizicky EM, Weissman JS, Koller D - Mol. Syst. Biol. (2014)

Comparison between codon translation rates in wild-type and mutantsCorrelation between estimated codon translation rates in wild-type versus mutant for the three mutant samples (the manipulated codon is highlighted in red). Rates are normalized by the minimum one in each sample. Pearson correlations are nearly exact, indicating that the mutant rates are generally unaffected.Source data are available online for this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300493&req=5

fig03: Comparison between codon translation rates in wild-type and mutantsCorrelation between estimated codon translation rates in wild-type versus mutant for the three mutant samples (the manipulated codon is highlighted in red). Rates are normalized by the minimum one in each sample. Pearson correlations are nearly exact, indicating that the mutant rates are generally unaffected.Source data are available online for this figure.
Mentions: Our analysis of elongation rates on endogenous mRNAs in the context of the co-adapted cellular tRNA pool addresses the effects of codon usage in natural physiology, but may be confounded by this co-adaptation and cannot directly test the causal links between various correlated mRNA features. To measure the effect of tRNA abundance on codon translation rate directly, we created three mutant yeast species to test whether (i) tRNA overexpression speeds up translation, (ii) the tRNA body itself causes the tRNA-dependent rate effect observed in other studies, or (iii) depletion of tRNA slows down ribosomes. In our first mutant, AGG-OE, the tRNA recognizing AGG (namely tRNAArg(CCU)) was overexpressed on a high-copy plasmid; in mutant AGG-QC, the body sequence of the tRNA recognizing AGG was swapped with the body of a more preferred tRNA (as measured by tAI); and in mutant ACA-K, three out of four copies of the tRNA recognizing ACA were deleted from the genome. The AGG mutants had a URA marker and were compared against a wild-type sample with a URA plasmid (see Materials and Methods). For ACA-K, we checked that the abundance of the tRNA for ACA (namely tRNAThr(UGU)) did decrease to about 30% of wild-type (Supplementary Table S1). In the AGG-OE mutant, we measured the amount of total and aminoacylated tRNA for tRNAArg(CCU) (see Materials and Methods) and verified that the tRNA was overexpressed by 13.8-fold (±0.4), based on an analysis of two independently derived RNA samples, and remained charged at a level similar to wild-type (87%) (Supplementary Fig S2). For the AGG-QC mutant, we similarly verified that the amount of charged tRNAArg(CCU) was similar to wild-type (Supplementary Fig S2). We generated ribosome profiling data and ran our model on these mutants to test whether AGG codons are translated faster in AGG-OE and AGG-QC and whether ACA codons are translated slower in ACA-K. We observe no significant change in the elongation rates of the affected codon in any of the three mutants compared to wild-type (Fig3, Supplementary Fig S3); the overall correlation between ACA-K and wild-type is not as tight as for other mutants, but this is due to changes affecting all codons, not only ACA. We verified the result by inspecting the footprint counts at the perturbed codon relative to adjacent counts in the mutants compared to wild-type and saw no unusual increase or decrease (Supplementary Fig S4). One prevailing hypothesis (Welch et al, 2009) is that the amount of charged as opposed to total tRNA is the true predictor of codon elongation; our measurements of aminoacylated tRNA suggest that these levels were manipulated as expected and that this is not a confounding factor in the mutant samples. Hence, our results suggest that several-fold changes in tRNA abundance do not affect ribosome dwell time.

Bottom Line: We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast.Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start.We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation.

View Article: PubMed Central - PubMed

Affiliation: Computer Science Department, Stanford University, Stanford, CA, USA cpop@cs.stanford.edu.

No MeSH data available.