1. Biochemistry and Chemical Biology
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Measurement of average decoding rates of the 61 sense codons in vivo

  1. Justin Gardin
  2. Rukhsana Yeasmin
  3. Alisa Yurovsky
  4. Ying Cai
  5. Steve Skiena
  6. Bruce Futcher  Is a corresponding author
  1. Stony Brook University, United States
Research Article
Cite this article as: eLife 2014;3:e03735 doi: 10.7554/eLife.03735
8 figures, 4 tables, 2 data sets and 4 additional files

Figures

Two ribosome profiles of the TDH1 gene.

Top profile is from the data of Ingolia et al., 2009; bottom profile is from the SC-lys dataset (‘Materials and methods’). The first (leftmost) peak in the profiles is at the ATG start codon; it may differ in relative height because the SC-lys dataset was generated using flash-freezing.

https://doi.org/10.7554/eLife.03735.003
Validation for ribosome residence time analysis.

(A) Simulated data, negative control. Real footprint data from the SC-lys dataset were randomly assigned to codons, and RRT analysis was carried out. A flat line with an RRT value of 1 indicates no signal. (B) Simulated data, positive control. A dataset of 2 million simulated reads was generated but biased to give more reads over the codon AAA at position 6. (C) Real data, negative control. RNA-seq data from naked fragments of RNA 30 nucleotides long, processed as if for ribosome profiling, were analyzed. (D) Real data, positive control. Real ribosome footprinting data from Li et al. were analyzed (Li et al., 2012). In this experiment, E. coli were starved for serine. Note that the highest Ser peak is for TCA, which is the rarest Ser codon in E. coli, and the lowest Ser peak is for AGC, which is the most common Ser codon in E. coli. High values at position 9 as well as 8 may indicate that the A-site may be at position 8 in some fragments and position 9 in others.

https://doi.org/10.7554/eLife.03735.004
Principle of ribosome residence time analysis.

The ribosome protects a 30 nt ‘footprint’ of RNA centered around the A, P, and E sites (positions 6, 5, and 4). The rare Leu codon CTC has a high RRT at position 6, which is likely the A-site.

https://doi.org/10.7554/eLife.03735.006
Results of Ribosome Residence Time analysis.

(A) The pattern of RRTs for all codons at all positions. Most peaks are at position 6, with some at position 5. (B) The RRTs for the six leucine codons. CTC has the highest RRT of any codon at position 6. (C) The RRTs for the four threonine codons. ACC has the lowest RRT of any codon at position 6. (D) The RRTs for the four proline codons. Proline has peaks at position 5, the P-site, as well as at position 6.

https://doi.org/10.7554/eLife.03735.007
Correlation of ribosome residence times with codon properties.

(A) Correlation of RRT with codon usage. RRT is plotted against the frequency of each codon per 1000 codons. (B) Correlation of RRT with the GC content of each codon. The codons were divided into quartiles by RRT (Fastest–Slowest), and the GC content of those ∼15 codons is shown in a violin plot.

https://doi.org/10.7554/eLife.03735.008
Analysis of ProPro dipeptides.

(A) RRT analysis of windows containing no ProPro dipeptides. (B) RRT analysis of windows containing ProPro dipeptides.

https://doi.org/10.7554/eLife.03735.010
RRT analysis of short footprints from anisomycin treatment.

The short, seven-codon footprints from anisomycin treatment (dataset 1b) from Lareau et al. (2014) were analyzed for RRT. All 61 sense codons are shown; codons for selected amino acids are color-coded by amino acid. Position along the footprint is shown on the x-axis.

https://doi.org/10.7554/eLife.03735.012
Short footprints are amino acid-specific; long footprints are codon-specific.

For the set of codons corresponding to each amino acid (x-axis), a test was done to see if all the codons behaved similarly or not. For the short footprints (left, panel A), p-values (y-axis) are generally small, showing that each codon for a particular amino acid behaves similarly (‘Materials and methods’). For the long footprints (right, panel B), p-values are generally large, showing that the codons for each particular amino acid behave differently (‘Materials and methods’).

https://doi.org/10.7554/eLife.03735.014

Tables

Table 1

Top ten RRTs at position 8 in E. coli starved for serine

https://doi.org/10.7554/eLife.03735.005
CodonAAUsageRRT
TCASer8.11.98
TCCSer9.01.90
TCGSer8.81.73
TCTSer8.71.71
AGTSer9.41.57
ATAIle5.51.42
AGCSer16.01.25
ATTIle29.71.18
CCTPro7.21.15
CCAPro8.41.13
Table 2

Ribosome residence time at position 6 (A) and 5 (B)

https://doi.org/10.7554/eLife.03735.009
A
CodonAAUsageRRTp value
CTCLeu5.41.89*0.0001
CCCPro6.81.71*0.0001
GGGGly61.61*0.0001
AGGArg9.21.59*0.0001
ATAIle17.81.57*0.0001
GGAGly10.91.56*0.0001
TGGTrp10.41.53*0.0001
GTGVal10.81.52*0.0001
CGCArg2.61.45*0.0001
CGAArg31.45*0.0008
CGGArg1.71.44*0.0010
TCGSer8.61.43*0.0001
CCAPro18.31.38*0.0001
ACAThr17.81.35*0.0001
CCGPro5.31.31*0.0001
GTAVal11.81.31*0.0001
GCAAla16.21.28*0.0001
CCTPro13.51.27*0.0001
TCASer18.71.26*0.0001
TACTyr14.81.25*0.0001
TATTyr18.81.25*0.0001
GAGGlu19.21.25*0.0001
CTALeu13.41.25*0.0001
CTTLeu12.31.24*0.0001
TGCCys4.81.23*0.0001
GGCGly9.81.22*0.0001
CAGGln12.11.15*0.0002
ACGThr81.120.0069
AGTSer14.21.100.0060
AGCSer9.81.090.0213
CACHis7.81.080.0098
TTTPhe26.11.050.0529
GAAGlu45.61.040.0538
AGAArg21.31.010.3014
TTCPhe18.41.000.4955
GCGAla6.20.990.4650
TCCSer14.20.990.3341
TTALeu26.20.990.3166
TCCSer23.50.980.2249
CATHis13.60.930.0188
GGTGly23.90.93*0.0003
ATGMet20.90.920.0027
ATTIle30.10.92*0.0005
TTGLeu27.20.92*0.0001
CTGLeu10.50.920.0139
AATAsn35.70.88*0.0001
AAALys41.90.88*0.0003
CGTArg6.40.87*0.0002
CAAGln27.30.87*0.0001
GCCAla12.60.86*0.0001
GACAsp20.20.85*0.0001
TGTCys8.10.81*0.0001
GCTAla21.20.81*0.0001
ATCIle17.20.80*0.0001
ACTThr20.30.78*0.0001
GATAsp37.60.76*0.0001
AACAsn24.80.76*0.0001
GTTVal22.10.75*0.0001
GTCVal11.80.75*0.0001
AAGLys30.80.74*0.0001
ACCThr12.70.70*0.0001
B
CodonAAUsageRRTp value
CCTPro13.51.80*0.0001
CCCPro6.81.48*0.0001
CCAPro18.31.48*0.0001
AATAsn35.71.39*0.0001
CGCArg1.71.340.0070
CCGPro5.31.30*0.0001
  1. A. Usage of each codon per 1000 codons and the Ribosome Residence Time (RRT) at position 6 (the A-site of the ribosome). The p-value for a difference between the calculated RRT value and an RRT value of 1 is shown. p-values less than or equal to 0.001 are marked with an asterisk. B. As for A, but for the six highest values at position 5 (the P-site).

Table 3

Correlations between experiments

https://doi.org/10.7554/eLife.03735.011
YPD1-HisYPD2Ingo.
-Lys0.800.350.760.22
YPD10.530.960.55
-His0.580.37
YPD20.53
  1. The pairwise Spearman correlations between the RRT values at position 6 are shown for five independent experiments, where the experiments are named YPD1, YPD2, SC-Lys, SC-His, and Ingolia. The SC-Lys and SC-His experiments were carried out by JG, and used flash-freezing as the initial method for stopping ribosome movement. The YPD1 and YPD2 experiments were carried out by YC (Cai and Futcher, 2013), and used addition of ice and cycloheximide to the culture as the initial method for stopping ribosome movement. The ‘Ingo’ experiment was that carried out by Ingolia et al. (2009). Further details are given in ‘Materials and methods’. Complete RRT values for each position in each experiment are provided in Supplementary file 1.

Table 4

Top 10 RRTs at positions 3 through 6 of the anisomycin-generated short footprints

https://doi.org/10.7554/eLife.03735.013
Pos 3Pos 4Pos 5Pos 6
Gly GGG 2.64Pro CCC 2.36Leu TTA 2.75Arg CGA 3.72
Gly GGC 2.52Pro CCA 2.34Leu CTC 2.73Arg CGG 3.50
Gly GGT 2.36Met ATG 2.25Val GTA 2.43Pro CCG 2.74
Gly GGA 2.32Pro CCT 2.17Leu CTA 2.36Lys AAA 2.59
Asp GAC 1.80Ala GCC 2.13Leu TTG 2.29Lys AAG 2.49
Ala GCC 1.79Phe TTC 2.03Val GTG 2.21Arg CGC 2.46
Ala GCA 1.70Ala GCA 2.01Leu CTT 2.16Arg CGT 2.34
Ala GCT 1.65Ala GCT 1.98Val GTC 2.12Arg AGG 2.32
Ala GCG 1.59Tyr TAC 1.98Val GTT 2.11Arg AGA 2.21
Blu GAG 1.58Ser TCC 1.97Ile ATA 2.03Asp GAT 2.12

Data availability

The following data sets were generated
    1. Gardin
    et al. (2014)
    Measurement of average decoding rates of the 61 sense codons in vivo
    ID SRP044053. NCBI SRA database.
The following previously published data sets were used
    1. Ying Cai
    (2013) Ribosome profiling of whi3 mutant yeast
    Publicly available at NCBI Gene Expression Omnibus.

Additional files

Supplementary file 1

Complete Ribosome Residence Times for each codon at each of the 10 possible codon positions in a 30 nt (or, for Ingolia data, 24 nt) ribosome footprint. Each Excel spreadsheet is based on data from an independent biological experiment. Four of these experiments were done during the course of this work, two experiments by JG and two experiments by YC, while the fifth experiment was published by Ingolia et al. (2009). (A) Ribosome Residence Time analysis for all codons from the SC-lys expt. (B) Ribosome Residence Time analysis from the YPD1(WT) expt. (C) Ribosome Residence Time analysis from the YPD2 (whi3) expt. (D) Ribosome Residence Time analysis from the SC-his expt. (E) Ribosome Residence Time analysis from the Ingolia expt.

https://doi.org/10.7554/eLife.03735.015
Supplementary file 2

Complete Ribosome Residence Times for each codon at each of the 7 possible codon positions in a 21 nt ribosome footprint. Each Table is based on one of the three anisomycin datasets of Lareau et al. (2014). (A) RRT for short footprints; aniso2 dataset. (B) RRT for short footprints; aniso1B dataset. (C) RRT for short footprints; aniso1A dataset.

https://doi.org/10.7554/eLife.03735.016
Source code 1

Source code 1 is a plain text file containing stage 1 of the Perl code for Ribosome Residence Time analysis.

https://doi.org/10.7554/eLife.03735.017
Source code 2

Source code 2 is a plain text file containing stage 2 of the Perl code for Ribosome Residence Time analysis.

https://doi.org/10.7554/eLife.03735.018

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