1. Biochemistry and Chemical Biology
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A discriminator code–based DTD surveillance ensures faithful glycine delivery for protein biosynthesis in bacteria

  1. Santosh Kumar Kuncha
  2. Katta Suma
  3. Komal Ishwar Pawar
  4. Jotin Gogoi
  5. Satya Brata Routh
  6. Sambhavi Pottabathini
  7. Shobha P Kruparani
  8. Rajan Sankaranarayanan  Is a corresponding author
  1. CSIR–Centre for Cellular and Molecular Biology, India
  2. CSIR–CCMB Campus, India
Research Advance
Cite this article as: eLife 2018;7:e38232 doi: 10.7554/eLife.38232
4 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
tRNAGly and tRNAAla show discriminator base dichotomy in Bacteria.

(a) Clover leaf model of tRNA with the discriminator base highlighted in red. (b) Frequency distribution of tRNA acceptor stem elements across bacterial tRNAs, comparing and contrasting between tRNAGly and tRNAAla. Red circle indicates the discriminator base. (c) Distribution of the discriminator base in all tRNAs across the three domains of life. The instances where the discriminator base shows >90% conservation has been represented by the most frequent base. In the case of tRNAThr, U73 and A73 together represent >90% frequency of occurrence; A/U in Bacteria implies A73 is more abundant than U73, whereas U/A in Eukarya denotes U73 is more abundant than A73. Amino acids are color-coded on the basis of the class to which the corresponding synthetases belong: yellow, class I; blue, class II; orange, both class I and II. Discriminator base color-coded as follows: green, purine (A or G); red, pyrimidine (U or C); grey, purine and pyrimidine (A/U or U/A).

https://doi.org/10.7554/eLife.38232.002
Figure 1—figure supplement 1
Multiple sequence alignment of a few E. coli tRNAs.

The second position is highlighted with a blue star while the discriminator base is marked with a red circle.

https://doi.org/10.7554/eLife.38232.003
Figure 1—figure supplement 2
Graph showing percentage distribution of the discriminator base in all tRNAs across all bacteria.
https://doi.org/10.7554/eLife.38232.004
Discriminator base modulates DTD’s activity.

(a–e) Deacylation of Gly-tRNAGly and its mutants by various concentrations of EcDTD. (f) Deacylation of Gly-tRNAAla by various concentrations of EcDTD. Lines indicate exponential decay fits and error bars represent one standard deviation from the mean of at least three independent readings.

https://doi.org/10.7554/eLife.38232.006
Figure 2—source data 1

Biochemical data for EcDTD deacylations with Gly-tRNAGly/Ala (wild type and mutants).

https://doi.org/10.7554/eLife.38232.007
Figure 3 with 1 supplement
Discriminator base predominantly determines the fate of the substrate.

Deacylation of (a) Gly-tRNAGly and (b) Gly-tRNAGly(U73A) by EcDTD in the presence or absence of EF-Tu (* indicates the data points are connected through line). Deacylation of (c) Gly-(Mm)tRNAGly and (d) Gly-(Mm)tRNAAla by various concentrations of MmDTD. Deacylation of (e) Gly-tRNAGly and (f) Gly-tRNAGly(U73A) by various concentrations of MmDTD. Lines indicate exponential decay fit and error bars represent one standard deviation from the mean of at least three independent readings.

https://doi.org/10.7554/eLife.38232.008
Figure 3—source data 1

Biochemical data for EF-Tu protection assays and MmDTD deacylations.

https://doi.org/10.7554/eLife.38232.010
Figure 3—figure supplement 1
Northern blotting showing PfDTD overexpression using IPTG leads to depletion of Gly-tRNAGly while the inactive mutant of PfDTD (A112F) has no effect.

Tris pH nine is used as deacylation control.

https://doi.org/10.7554/eLife.38232.009
DTD avoids glycine misincorporation into proteins.

(a) GFP-based fluorescence reporter assay for visualizing alanine-to-glycine mistranslation, wherein the mutant GFP G67A (65TYA67) will fluoresce only when TYA is mistranslated to TYG. Microscopy images showing GFP fluorescence in E. coli at different concentrations of glycine supplementation (b) in the presence and (c) in the absence of DTD. The E. coli strain used is MG1655 with editing-defective AlaRS gene (i.e. ΔalaS) (Pawar et al., 2017). (d) Model showing N73 dichotomy in bacterial tRNAGly and tRNAAla, enabling protection of the cognate Gly-tRNAGly (both proteinogenic and non-proteinogenic) predominantly by U73, while effecting efficient removal of the non-cognate Gly-tRNAAla (having A73 and G3•U70) to prevent alanine-to-glycine mistranslation.

https://doi.org/10.7554/eLife.38232.011

Tables

Table 1
Table showing the number of tRNAs having a particular discriminator base for all tRNAs across bacteria.
https://doi.org/10.7554/eLife.38232.005
tRNAXA73G73C73U73Total
Ala136822969136846
Ile61982283362016
Leu2596241395259651
Lys12306412240510123736
Met207181122531868209314
Phe68315202468341
Pro110670913110683
Tyr80595565080656
Val1872501073187270
Arg1198191030523210151233054
Glu294695476484684287
Asn20129816641129883
Asp1712155613121577
Gln78710240562250105448
Ser133619269763119197158
Trp55438702354415
Thr131288349528854160271
Cys5285695656971
Gly239114231855232109
His20044560521758077
Total bacterial tRNAs:2,671,763

Data availability

Biochemical data is available as a source data file. All other data are included in the manuscript and supporting files.

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