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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 in absence of the S9 tail alone or in combination with deletion of rsmD with various initiation codons using the native initiator tRNA (tRNA). A tail ending with SKR in the box on the top right indicates ribosomal protein S9. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmD, S9Δ3, and S9Δ3 ΔrsmD derivatives are shown. For reference, the average CAT activities in SA strain (C, taken as 1) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7838 ± 610, 3198 ± 378, 3788 ± 334, 327 ± 15, 313 ± 19, 346 ± 34, 631 ± 71.6 and 78 ± 9 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
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gkt175-F5: Initiation in absence of the S9 tail alone or in combination with deletion of rsmD with various initiation codons using the native initiator tRNA (tRNA). A tail ending with SKR in the box on the top right indicates ribosomal protein S9. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmD, S9Δ3, and S9Δ3 ΔrsmD derivatives are shown. For reference, the average CAT activities in SA strain (C, taken as 1) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7838 ± 610, 3198 ± 378, 3788 ± 334, 327 ± 15, 313 ± 19, 346 ± 34, 631 ± 71.6 and 78 ± 9 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Mentions: Alternate initiation codons GUG, UUG and CUG with a mismatch at the first codon position are not uncommon in mRNAs and are decoded by the native tRNA. CAT reporters that initiate from these codons showed that although the S9 tail deletion did not impact initiation from AUG, it led to a decrease in initiation from GUG, UUG and CUG (Figure 5). In the absence of the S9 tail, the mismatched codon:anticodon pairs may be less stable, and the tail may be required to maintain interactions between the anticodon and the codon with first base wobble/mismatch. Initiation from ACG; and AUA, AUC, and AUU with the native tRNA (mismatches at the second or the third positions of the codon) showed that these activities remained unchanged in the S9Δ3 strain (Figure 5).Figure 5.


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 in absence of the S9 tail alone or in combination with deletion of rsmD with various initiation codons using the native initiator tRNA (tRNA). A tail ending with SKR in the box on the top right indicates ribosomal protein S9. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmD, S9Δ3, and S9Δ3 ΔrsmD derivatives are shown. For reference, the average CAT activities in SA strain (C, taken as 1) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7838 ± 610, 3198 ± 378, 3788 ± 334, 327 ± 15, 313 ± 19, 346 ± 34, 631 ± 71.6 and 78 ± 9 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-F5: Initiation in absence of the S9 tail alone or in combination with deletion of rsmD with various initiation codons using the native initiator tRNA (tRNA). A tail ending with SKR in the box on the top right indicates ribosomal protein S9. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmD, S9Δ3, and S9Δ3 ΔrsmD derivatives are shown. For reference, the average CAT activities in SA strain (C, taken as 1) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7838 ± 610, 3198 ± 378, 3788 ± 334, 327 ± 15, 313 ± 19, 346 ± 34, 631 ± 71.6 and 78 ± 9 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
Mentions: Alternate initiation codons GUG, UUG and CUG with a mismatch at the first codon position are not uncommon in mRNAs and are decoded by the native tRNA. CAT reporters that initiate from these codons showed that although the S9 tail deletion did not impact initiation from AUG, it led to a decrease in initiation from GUG, UUG and CUG (Figure 5). In the absence of the S9 tail, the mismatched codon:anticodon pairs may be less stable, and the tail may be required to maintain interactions between the anticodon and the codon with first base wobble/mismatch. Initiation from ACG; and AUA, AUC, and AUU with the native tRNA (mismatches at the second or the third positions of the codon) showed that these activities remained unchanged in the S9Δ3 strain (Figure 5).Figure 5.

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