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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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Pictorial representation of the dynamically stable H-bonds on the tRNA–mRNA and S9 complex from PDB accession number 2J00 in AUG:CAU (panel i), CAC:GUG (panel ii), CAU:GUG (panel iii) and AUC:GAU (panel iv) codon:anticodon pairs in the presence and absence of the S9 tail, respectively. The H-bonds present in 70% or more snapshots are depicted by blue line, whereas those in 60–70%, 50–60% and 40–50% of the snapshots are depicted by cyan, yellow and red lines, respectively. The tRNAfMet and mRNA backbones are depicted as deep green wires, and the residues of interest (both amino acids and nucleotides), participating in the H-bonds, are shown in stick representation.
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gkt175-F7: Pictorial representation of the dynamically stable H-bonds on the tRNA–mRNA and S9 complex from PDB accession number 2J00 in AUG:CAU (panel i), CAC:GUG (panel ii), CAU:GUG (panel iii) and AUC:GAU (panel iv) codon:anticodon pairs in the presence and absence of the S9 tail, respectively. The H-bonds present in 70% or more snapshots are depicted by blue line, whereas those in 60–70%, 50–60% and 40–50% of the snapshots are depicted by cyan, yellow and red lines, respectively. The tRNAfMet and mRNA backbones are depicted as deep green wires, and the residues of interest (both amino acids and nucleotides), participating in the H-bonds, are shown in stick representation.

Mentions: The MD simulations of AUG:CAU, CAC:GUG and CAU:GUG codon:anticodon pairs with or without the S9 tail did not exhibit any drastic conformational changes at the backbone level of tRNAfMet. However, a rigorous H-bond analysis along the trajectory provided excellent means of capturing subtle conformational re-orientations, taking into account the minor rewiring at the side-chain level. The AUG:CAU pair exhibits a dynamically stable H-bond (indicated by blue line, Figure 7, panel i) with the S9 tail, which exists in >70% of the snapshots. Another H-bond (indicated by yellow line) was observed in fewer (∼57%) snapshots. Of these, the one indicated in blue was also reported in crystal structure of the initiator tRNA ribosome complex (4). The deletion of the S9 tail (leading to loss of these weak H-bonds) is expected to show no major effects on the interaction of native anticodon (CAU) with the AUG initiation codon. In the CAC:GUG codon:anticodon pair, substitution of C34 anticodon position by G34 affects subtle conformational variations during dynamics, which induced enhanced H-bonding of the tRNA with the S9 tail (Figure 7, panel ii). These H-bonds arrest the intrinsic dynamics and have a freezing effect on the tRNA, which is released on the S9 tail deletion. Thus, the S9 tail deletion is proposed to allow the tRNA regain the conformational dynamics important for efficient recognition of the CAC initiation codon. As shown in Figure 7, panel iii, for the CAU:GUG codon:anticodon pair, along with the mutation at the 34 position of the anticodon, a GU wobble (at the third position of the codon) is also incorporated. Here, we observe not only the formation of additional H-bonds (i.e. enhanced tRNA:S9 interaction) but also loss of a non-Watson and Crick H-bond between the third G:U pair in the presence of the S9 tail. On deletion of the S9 tail, the lost H-bond between the G:U as also the dynamical properties of the system are regained, thus facilitating codon:anticodon pairing. These observations are in good agreement with the in vivo data showing an increase in initiation from CAC and CAU on S9 tail deletion (Figure 4).Figure 7.


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)

Pictorial representation of the dynamically stable H-bonds on the tRNA–mRNA and S9 complex from PDB accession number 2J00 in AUG:CAU (panel i), CAC:GUG (panel ii), CAU:GUG (panel iii) and AUC:GAU (panel iv) codon:anticodon pairs in the presence and absence of the S9 tail, respectively. The H-bonds present in 70% or more snapshots are depicted by blue line, whereas those in 60–70%, 50–60% and 40–50% of the snapshots are depicted by cyan, yellow and red lines, respectively. The tRNAfMet and mRNA backbones are depicted as deep green wires, and the residues of interest (both amino acids and nucleotides), participating in the H-bonds, are shown in stick representation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt175-F7: Pictorial representation of the dynamically stable H-bonds on the tRNA–mRNA and S9 complex from PDB accession number 2J00 in AUG:CAU (panel i), CAC:GUG (panel ii), CAU:GUG (panel iii) and AUC:GAU (panel iv) codon:anticodon pairs in the presence and absence of the S9 tail, respectively. The H-bonds present in 70% or more snapshots are depicted by blue line, whereas those in 60–70%, 50–60% and 40–50% of the snapshots are depicted by cyan, yellow and red lines, respectively. The tRNAfMet and mRNA backbones are depicted as deep green wires, and the residues of interest (both amino acids and nucleotides), participating in the H-bonds, are shown in stick representation.
Mentions: The MD simulations of AUG:CAU, CAC:GUG and CAU:GUG codon:anticodon pairs with or without the S9 tail did not exhibit any drastic conformational changes at the backbone level of tRNAfMet. However, a rigorous H-bond analysis along the trajectory provided excellent means of capturing subtle conformational re-orientations, taking into account the minor rewiring at the side-chain level. The AUG:CAU pair exhibits a dynamically stable H-bond (indicated by blue line, Figure 7, panel i) with the S9 tail, which exists in >70% of the snapshots. Another H-bond (indicated by yellow line) was observed in fewer (∼57%) snapshots. Of these, the one indicated in blue was also reported in crystal structure of the initiator tRNA ribosome complex (4). The deletion of the S9 tail (leading to loss of these weak H-bonds) is expected to show no major effects on the interaction of native anticodon (CAU) with the AUG initiation codon. In the CAC:GUG codon:anticodon pair, substitution of C34 anticodon position by G34 affects subtle conformational variations during dynamics, which induced enhanced H-bonding of the tRNA with the S9 tail (Figure 7, panel ii). These H-bonds arrest the intrinsic dynamics and have a freezing effect on the tRNA, which is released on the S9 tail deletion. Thus, the S9 tail deletion is proposed to allow the tRNA regain the conformational dynamics important for efficient recognition of the CAC initiation codon. As shown in Figure 7, panel iii, for the CAU:GUG codon:anticodon pair, along with the mutation at the 34 position of the anticodon, a GU wobble (at the third position of the codon) is also incorporated. Here, we observe not only the formation of additional H-bonds (i.e. enhanced tRNA:S9 interaction) but also loss of a non-Watson and Crick H-bond between the third G:U pair in the presence of the S9 tail. On deletion of the S9 tail, the lost H-bond between the G:U as also the dynamical properties of the system are regained, thus facilitating codon:anticodon pairing. These observations are in good agreement with the in vivo data showing an increase in initiation from CAC and CAU on S9 tail deletion (Figure 4).Figure 7.

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