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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 activities of the native tRNA from various codons. Star symbols in the box 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 the SA control (C) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7314 ± 923, 3629 ± 492, 4019 ± 1000, 906 ± 132, 238 ± 33, 301 ± 32, 352.5 ± 38 and 48 ± 15 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
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gkt175-F3: Initiation activities of the native tRNA from various codons. Star symbols in the box 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 the SA control (C) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7314 ± 923, 3629 ± 492, 4019 ± 1000, 906 ± 132, 238 ± 33, 301 ± 32, 352.5 ± 38 and 48 ± 15 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Mentions: Deletion of rsmD resulted in increased initiation with the CAU:GUG pair (U:G mismatch at the third position of codon) to a level (>3-fold) that was higher than that seen for the perfectly matched pair CAC:GUG (∼2-fold) (Figure 2B). This led us to investigate whether G966 methylation discriminated against the third position (of the initiation codon) wobble/mismatch. To address this, initiation from various canonical codons AUG, GUG, UUG (28) and CUG (29) and non-canonical codons AUU (2), AUA (30), AUC and ACG with the native initiator tRNA (Supplementary Table S4) was investigated. As seen in Figure 3, on deletion of rsmD, initiation from AUA (third base mismatch with tRNA) was at an advantage (∼2-fold), but initiation from the remaining codons was not impacted significantly. Also, deletion of rsmB (alone or in combination with rsmD) did not have much impact.Figure 3.


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 activities of the native tRNA from various codons. Star symbols in the box 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 the SA control (C) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7314 ± 923, 3629 ± 492, 4019 ± 1000, 906 ± 132, 238 ± 33, 301 ± 32, 352.5 ± 38 and 48 ± 15 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

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

gkt175-F3: Initiation activities of the native tRNA from various codons. Star symbols in the box 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 the SA control (C) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7314 ± 923, 3629 ± 492, 4019 ± 1000, 906 ± 132, 238 ± 33, 301 ± 32, 352.5 ± 38 and 48 ± 15 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
Mentions: Deletion of rsmD resulted in increased initiation with the CAU:GUG pair (U:G mismatch at the third position of codon) to a level (>3-fold) that was higher than that seen for the perfectly matched pair CAC:GUG (∼2-fold) (Figure 2B). This led us to investigate whether G966 methylation discriminated against the third position (of the initiation codon) wobble/mismatch. To address this, initiation from various canonical codons AUG, GUG, UUG (28) and CUG (29) and non-canonical codons AUU (2), AUA (30), AUC and ACG with the native initiator tRNA (Supplementary Table S4) was investigated. As seen in Figure 3, on deletion of rsmD, initiation from AUA (third base mismatch with tRNA) was at an advantage (∼2-fold), but initiation from the remaining codons was not impacted significantly. Also, deletion of rsmB (alone or in combination with rsmD) did not have much impact.Figure 3.

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