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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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P-site model showing interaction of CAU anticodon (C34, A35 and U36) of tRNAfMet with AUG initiation codon (A1, U2 and G3). Contacts of G966, C967 and the S9 tail are shown. Spheres associated with G966 and C967 indicate methyl groups at their positions 2 and 5, respectively. Lys127 of S9 tail contacts C34 of the anticodon and is in proximity to G966 and C967. The model was generated with PyMOL v1.3r1 using PDB accession number 2J00 (4).
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gkt175-F1: P-site model showing interaction of CAU anticodon (C34, A35 and U36) of tRNAfMet with AUG initiation codon (A1, U2 and G3). Contacts of G966, C967 and the S9 tail are shown. Spheres associated with G966 and C967 indicate methyl groups at their positions 2 and 5, respectively. Lys127 of S9 tail contacts C34 of the anticodon and is in proximity to G966 and C967. The model was generated with PyMOL v1.3r1 using PDB accession number 2J00 (4).

Mentions: High-resolution co-crystal structures of the 70S ribosome bound with tRNAfMet in the P-site (together with tRNAPhe bound in the A- and E-, sites) and an mRNA (4,5) yielded insights into the distinctive features of the P-site. A methylated nucleoside of the 16S rRNA, G966, stacks against the ribose of the first base of the anticodon (C34) of the tRNAfMet. The residue C967, also a methylated nucleoside, lies next to G966 and is also in proximity to tRNAfMet (Figure 1). The G966 and C967 methylations are carried out by the specific methyltransferases, RsmD and RsmB, respectively (6,7). The structural data (4) have also revealed that the tail of S9 protein extends into the P-site and contacts tRNAfMet at positions 33 and 34 (Figure 1). The C-terminal tail sequence of the S9 protein is phylogenetically conserved (8). And, although modification of residue 966 is also conserved, neither the identity of the residue nor the nature of the modification is conserved (9,10). The residues G966 and C967 lie in the 970 loop, which forms helix 31, and mutation of either has been reported to result in a slight increase (107–127%) in production of a reporter protein (11). The C967 methylation is conserved in bacteria, and the two methylated nucleosides are also reported to contact the S9 tail via the backbone interactions (12,13), thus forming a network of interactions with tRNAfMet in the P-site. In recent years, roles of post-transcriptionally modified nucleosides have been studied by several groups, and methylated nucleosides have been shown to impact fidelity of initiation (14), ribosome recycling (15,16), ribosome biogenesis (17) and response to nascent peptide (18).Figure 1.


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)

P-site model showing interaction of CAU anticodon (C34, A35 and U36) of tRNAfMet with AUG initiation codon (A1, U2 and G3). Contacts of G966, C967 and the S9 tail are shown. Spheres associated with G966 and C967 indicate methyl groups at their positions 2 and 5, respectively. Lys127 of S9 tail contacts C34 of the anticodon and is in proximity to G966 and C967. The model was generated with PyMOL v1.3r1 using PDB accession number 2J00 (4).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt175-F1: P-site model showing interaction of CAU anticodon (C34, A35 and U36) of tRNAfMet with AUG initiation codon (A1, U2 and G3). Contacts of G966, C967 and the S9 tail are shown. Spheres associated with G966 and C967 indicate methyl groups at their positions 2 and 5, respectively. Lys127 of S9 tail contacts C34 of the anticodon and is in proximity to G966 and C967. The model was generated with PyMOL v1.3r1 using PDB accession number 2J00 (4).
Mentions: High-resolution co-crystal structures of the 70S ribosome bound with tRNAfMet in the P-site (together with tRNAPhe bound in the A- and E-, sites) and an mRNA (4,5) yielded insights into the distinctive features of the P-site. A methylated nucleoside of the 16S rRNA, G966, stacks against the ribose of the first base of the anticodon (C34) of the tRNAfMet. The residue C967, also a methylated nucleoside, lies next to G966 and is also in proximity to tRNAfMet (Figure 1). The G966 and C967 methylations are carried out by the specific methyltransferases, RsmD and RsmB, respectively (6,7). The structural data (4) have also revealed that the tail of S9 protein extends into the P-site and contacts tRNAfMet at positions 33 and 34 (Figure 1). The C-terminal tail sequence of the S9 protein is phylogenetically conserved (8). And, although modification of residue 966 is also conserved, neither the identity of the residue nor the nature of the modification is conserved (9,10). The residues G966 and C967 lie in the 970 loop, which forms helix 31, and mutation of either has been reported to result in a slight increase (107–127%) in production of a reporter protein (11). The C967 methylation is conserved in bacteria, and the two methylated nucleosides are also reported to contact the S9 tail via the backbone interactions (12,13), thus forming a network of interactions with tRNAfMet in the P-site. In recent years, roles of post-transcriptionally modified nucleosides have been studied by several groups, and methylated nucleosides have been shown to impact fidelity of initiation (14), ribosome recycling (15,16), ribosome biogenesis (17) and response to nascent peptide (18).Figure 1.

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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