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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 with CAC:GUG (panel i), CAU:GUG (panel ii), UAC:GUA (panel iii), AUC:GAU (panel iv), AUG:CAU (panel v) and UAG:CUA (panel vi) codon:anticodon pairs. A tail ending in SKR 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 (C) for the CAC:GUG, CAU:GUG, UAC:GUA, AUC:GAU, AUG:CAU and UAG:CUA constructs were 1236 ± 151, 590 ± 103, 241 ± 26, 12266 ± 2866, 7838 ± 610 and 5454 ± 682 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
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gkt175-F4: Initiation with CAC:GUG (panel i), CAU:GUG (panel ii), UAC:GUA (panel iii), AUC:GAU (panel iv), AUG:CAU (panel v) and UAG:CUA (panel vi) codon:anticodon pairs. A tail ending in SKR 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 (C) for the CAC:GUG, CAU:GUG, UAC:GUA, AUC:GAU, AUG:CAU and UAG:CUA constructs were 1236 ± 151, 590 ± 103, 241 ± 26, 12266 ± 2866, 7838 ± 610 and 5454 ± 682 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Mentions: E. coli SA and its derivatives were transformed with pCATCACmetYGUG, pCATCAUmetYGUG, pCATAUG or pCATUAGmetYCUA and used to monitor initiation (Figure 4). Initiation from the unnatural codons, CAC and CAU, using tRNA showed ∼3- to 4-fold increase in initiation in S9Δ3 and S9Δ3 ΔrsmD strains (panels i and ii). However, initiation from AUG using tRNA or from UAG using a near natural tRNA (panels v and vi) showed no significant changes. Combining rsmD deletion with S9Δ3 (ΔrsmD S9Δ3) did not increase initiation any significantly (Figure 4).Figure 4.


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 with CAC:GUG (panel i), CAU:GUG (panel ii), UAC:GUA (panel iii), AUC:GAU (panel iv), AUG:CAU (panel v) and UAG:CUA (panel vi) codon:anticodon pairs. A tail ending in SKR 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 (C) for the CAC:GUG, CAU:GUG, UAC:GUA, AUC:GAU, AUG:CAU and UAG:CUA constructs were 1236 ± 151, 590 ± 103, 241 ± 26, 12266 ± 2866, 7838 ± 610 and 5454 ± 682 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-F4: Initiation with CAC:GUG (panel i), CAU:GUG (panel ii), UAC:GUA (panel iii), AUC:GAU (panel iv), AUG:CAU (panel v) and UAG:CUA (panel vi) codon:anticodon pairs. A tail ending in SKR 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 (C) for the CAC:GUG, CAU:GUG, UAC:GUA, AUC:GAU, AUG:CAU and UAG:CUA constructs were 1236 ± 151, 590 ± 103, 241 ± 26, 12266 ± 2866, 7838 ± 610 and 5454 ± 682 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
Mentions: E. coli SA and its derivatives were transformed with pCATCACmetYGUG, pCATCAUmetYGUG, pCATAUG or pCATUAGmetYCUA and used to monitor initiation (Figure 4). Initiation from the unnatural codons, CAC and CAU, using tRNA showed ∼3- to 4-fold increase in initiation in S9Δ3 and S9Δ3 ΔrsmD strains (panels i and ii). However, initiation from AUG using tRNA or from UAG using a near natural tRNA (panels v and vi) showed no significant changes. Combining rsmD deletion with S9Δ3 (ΔrsmD S9Δ3) did not increase initiation any significantly (Figure 4).Figure 4.

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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