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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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Effect of deletion of the S9 tail on initiation with the anticodon stem mutants of tRNA. Various mutations in the anticodon stem are as shown (A). Fold differences in the CAT activities with respect to SA (C, taken as 1) in its S9Δ3 derivatives are shown (B) for 1GC (AU, panel i), 1GC (GU, panel ii), 2GC (AU/GU, panel iii) and the 3GC mutant (panel iv). For reference, the average CAT activities in SA for the UAG:CUA/AU, UAG:CUA/GU, UAG:CUA/AUGU and UAG:CUA/3GC constructs were 2970 ± 900, 888 ± 145, 32 ± 6, 238 ± 33 and 88 ± 19 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
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gkt175-F6: Effect of deletion of the S9 tail on initiation with the anticodon stem mutants of tRNA. Various mutations in the anticodon stem are as shown (A). Fold differences in the CAT activities with respect to SA (C, taken as 1) in its S9Δ3 derivatives are shown (B) for 1GC (AU, panel i), 1GC (GU, panel ii), 2GC (AU/GU, panel iii) and the 3GC mutant (panel iv). For reference, the average CAT activities in SA for the UAG:CUA/AU, UAG:CUA/GU, UAG:CUA/AUGU and UAG:CUA/3GC constructs were 2970 ± 900, 888 ± 145, 32 ± 6, 238 ± 33 and 88 ± 19 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Mentions: The S9 tail is flexible, and we hypothesized that it may sense alterations in the conformation of the anticodon arm of the initiator tRNA. To investigate whether the S9 tail imposes any preference for the tRNAfMet via its highly conserved feature of the three consecutive GC (3GC) base pairs in the anticodon stem, we used tRNAfMet mutants (Figure 6A) possessing changes at the first, first and third, third or all 3GC base pairs (15,31). The CAT reporters were introduced into E. coli SA and its S9Δ3 derivative. As expected from our earlier observation (Figure 4, panel vi), the initiation activity of tRNA (with 3GC base pairs intact) from UAG did not change on deletion of the S9 tail. However, the initiation activities of the 2GC and the 3GC variants (A:U/G:U and 3GC; Figure 6B, panels iii and iv) showed an increase of >2-fold. The increases in initiation by the 1GC mutants, not unexpectedly, were subtle (Figure 6, panels i and ii).Figure 6.


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)

Effect of deletion of the S9 tail on initiation with the anticodon stem mutants of tRNA. Various mutations in the anticodon stem are as shown (A). Fold differences in the CAT activities with respect to SA (C, taken as 1) in its S9Δ3 derivatives are shown (B) for 1GC (AU, panel i), 1GC (GU, panel ii), 2GC (AU/GU, panel iii) and the 3GC mutant (panel iv). For reference, the average CAT activities in SA for the UAG:CUA/AU, UAG:CUA/GU, UAG:CUA/AUGU and UAG:CUA/3GC constructs were 2970 ± 900, 888 ± 145, 32 ± 6, 238 ± 33 and 88 ± 19 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-F6: Effect of deletion of the S9 tail on initiation with the anticodon stem mutants of tRNA. Various mutations in the anticodon stem are as shown (A). Fold differences in the CAT activities with respect to SA (C, taken as 1) in its S9Δ3 derivatives are shown (B) for 1GC (AU, panel i), 1GC (GU, panel ii), 2GC (AU/GU, panel iii) and the 3GC mutant (panel iv). For reference, the average CAT activities in SA for the UAG:CUA/AU, UAG:CUA/GU, UAG:CUA/AUGU and UAG:CUA/3GC constructs were 2970 ± 900, 888 ± 145, 32 ± 6, 238 ± 33 and 88 ± 19 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.
Mentions: The S9 tail is flexible, and we hypothesized that it may sense alterations in the conformation of the anticodon arm of the initiator tRNA. To investigate whether the S9 tail imposes any preference for the tRNAfMet via its highly conserved feature of the three consecutive GC (3GC) base pairs in the anticodon stem, we used tRNAfMet mutants (Figure 6A) possessing changes at the first, first and third, third or all 3GC base pairs (15,31). The CAT reporters were introduced into E. coli SA and its S9Δ3 derivative. As expected from our earlier observation (Figure 4, panel vi), the initiation activity of tRNA (with 3GC base pairs intact) from UAG did not change on deletion of the S9 tail. However, the initiation activities of the 2GC and the 3GC variants (A:U/G:U and 3GC; Figure 6B, panels iii and iv) showed an increase of >2-fold. The increases in initiation by the 1GC mutants, not unexpectedly, were subtle (Figure 6, panels i and ii).Figure 6.

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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