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Distinctive contributions of the ribosomal P-site elements m2G966, m5C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli.

Arora S, Bhamidimarri SP, Bhattacharyya M, Govindan A, Weber MH, Vishveshwara S, Varshney U - Nucleic Acids Res. (2013)

Bottom Line: Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold).Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold).These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(fMet(CAU) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(fMet(GUG); UAG with tRNA(fMet(CAU) ; UAC with tRNA(fMet(GUG) ; and AUC with tRNA(fMet(GUG) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(fMet(CAU) lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

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Initiation from CAC and CAU codons by tRNA (encoded by metYGUG) as assessed by growth on Cm plate (A). Saturated cultures of E. coli SA and its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives harbouring pCATCAUmetYGUG (sectors 1–4) or pCATCACmetYGUG (sectors 5–8) were streaked on Amp100 (left) or Amp100 Cm250 (right) and incubated at 37°C for ∼15 h. CAT assays (B) to assess initiation activities of CAC:GUG (panel i), CAU:GUG (panel ii), AUG:CAU (panel iii) and UAG:CUA (panel iv) codon:anticodon pairs. Star symbols in the boxes indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in SA (C) for the CAC:GUG, CAU:GUG, AUG:CAU and UAG:CUA codon:anticodon pairs were 1462 ± 374, 493 ± 64, 7314 ± 923 and 5246 ± 763 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
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gkt175-F2: Initiation from CAC and CAU codons by tRNA (encoded by metYGUG) as assessed by growth on Cm plate (A). Saturated cultures of E. coli SA and its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives harbouring pCATCAUmetYGUG (sectors 1–4) or pCATCACmetYGUG (sectors 5–8) were streaked on Amp100 (left) or Amp100 Cm250 (right) and incubated at 37°C for ∼15 h. CAT assays (B) to assess initiation activities of CAC:GUG (panel i), CAU:GUG (panel ii), AUG:CAU (panel iii) and UAG:CUA (panel iv) codon:anticodon pairs. Star symbols in the boxes indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in SA (C) for the CAC:GUG, CAU:GUG, AUG:CAU and UAG:CUA codon:anticodon pairs were 1462 ± 374, 493 ± 64, 7314 ± 923 and 5246 ± 763 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Mentions: The pCATCACmetYGUG and pCATCAUmetYGUG constructs were introduced into E. coli SA and its derivatives to measure initiation from CAC:GUG and CAU:GUG codon:anticodon pairs, respectively. Transformants were grown in LB with Amp and streaked on LB-agar containing 100 μg ml−1 Amp (Amp100) as control and on LB-agar Amp100 with varying concentrations of Cm (Amp100 Cm100−250). Representative plates with Amp100 or Amp100 Cm250 (Figure 2A) show that the pCATCAUmetYGUG supported the growth of the strains deleted for methyltransferases in singles (ΔrsmB or ΔrsmD) or double (ΔrsmB ΔrsmD) (sectors 2–4) but not of the parent (sector 1), suggesting better initiation by tRNA in the absence of methylations at positions 966 and/or 967. In the plate assay, the transformants harbouring pCATCACmetYGUG (sectors 5–8) did not show a perceivable difference in growth (because of higher level of initiation from the matched CAC codon). However, as revealed by the CAT assays (Figure 2B, panels i and ii), deletion of methyltransferases in singles or double resulted in increased initiation by tRNA (∼2- to 3.6-fold) over the SA parent (C) from both CAC and CAU codons (see also Supplementary Figure S6). Interestingly, as shown in Figure 2B (panels iii and iv), the deficiency of these methyltransferases showed no significant changes in initiation with AUG (decoded by native tRNA) or UAG (decoded by near native tRNA).Figure 2.


Distinctive contributions of the ribosomal P-site elements m2G966, m5C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli.

Arora S, Bhamidimarri SP, Bhattacharyya M, Govindan A, Weber MH, Vishveshwara S, Varshney U - Nucleic Acids Res. (2013)

Initiation from CAC and CAU codons by tRNA (encoded by metYGUG) as assessed by growth on Cm plate (A). Saturated cultures of E. coli SA and its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives harbouring pCATCAUmetYGUG (sectors 1–4) or pCATCACmetYGUG (sectors 5–8) were streaked on Amp100 (left) or Amp100 Cm250 (right) and incubated at 37°C for ∼15 h. CAT assays (B) to assess initiation activities of CAC:GUG (panel i), CAU:GUG (panel ii), AUG:CAU (panel iii) and UAG:CUA (panel iv) codon:anticodon pairs. Star symbols in the boxes indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in SA (C) for the CAC:GUG, CAU:GUG, AUG:CAU and UAG:CUA codon:anticodon pairs were 1462 ± 374, 493 ± 64, 7314 ± 923 and 5246 ± 763 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643588&req=5

gkt175-F2: Initiation from CAC and CAU codons by tRNA (encoded by metYGUG) as assessed by growth on Cm plate (A). Saturated cultures of E. coli SA and its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives harbouring pCATCAUmetYGUG (sectors 1–4) or pCATCACmetYGUG (sectors 5–8) were streaked on Amp100 (left) or Amp100 Cm250 (right) and incubated at 37°C for ∼15 h. CAT assays (B) to assess initiation activities of CAC:GUG (panel i), CAU:GUG (panel ii), AUG:CAU (panel iii) and UAG:CUA (panel iv) codon:anticodon pairs. Star symbols in the boxes indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in SA (C) for the CAC:GUG, CAU:GUG, AUG:CAU and UAG:CUA codon:anticodon pairs were 1462 ± 374, 493 ± 64, 7314 ± 923 and 5246 ± 763 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
Mentions: The pCATCACmetYGUG and pCATCAUmetYGUG constructs were introduced into E. coli SA and its derivatives to measure initiation from CAC:GUG and CAU:GUG codon:anticodon pairs, respectively. Transformants were grown in LB with Amp and streaked on LB-agar containing 100 μg ml−1 Amp (Amp100) as control and on LB-agar Amp100 with varying concentrations of Cm (Amp100 Cm100−250). Representative plates with Amp100 or Amp100 Cm250 (Figure 2A) show that the pCATCAUmetYGUG supported the growth of the strains deleted for methyltransferases in singles (ΔrsmB or ΔrsmD) or double (ΔrsmB ΔrsmD) (sectors 2–4) but not of the parent (sector 1), suggesting better initiation by tRNA in the absence of methylations at positions 966 and/or 967. In the plate assay, the transformants harbouring pCATCACmetYGUG (sectors 5–8) did not show a perceivable difference in growth (because of higher level of initiation from the matched CAC codon). However, as revealed by the CAT assays (Figure 2B, panels i and ii), deletion of methyltransferases in singles or double resulted in increased initiation by tRNA (∼2- to 3.6-fold) over the SA parent (C) from both CAC and CAU codons (see also Supplementary Figure S6). Interestingly, as shown in Figure 2B (panels iii and iv), the deficiency of these methyltransferases showed no significant changes in initiation with AUG (decoded by native tRNA) or UAG (decoded by near native tRNA).Figure 2.

Bottom Line: Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold).Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold).These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(fMet(CAU) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(fMet(GUG); UAG with tRNA(fMet(CAU) ; UAC with tRNA(fMet(GUG) ; and AUC with tRNA(fMet(GUG) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(fMet(CAU) lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

Show MeSH
Related in: MedlinePlus