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

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.

Figure 1.

P-site model showing interaction of CAU anticodon (C34, A35 and U36) of tRNA^fMet 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).

Figure 2.

Initiation from CAC and CAU codons by tRNA (encoded by metY_GUG) as assessed by growth on Cm plate (A). Saturated cultures of E. coli SA and its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives harbouring pCAT_CAUmetY_GUG (sectors 1–4) or pCAT_CACmetY_GUG (sectors 5–8) were streaked on Amp¹⁰⁰ (left) or Amp¹⁰⁰ Cm²⁵⁰ (right) and incubated at 37°C for ∼15 h. CAT assays (B) to assess initiation activities of CAC:GUG (panel i), CAU:GUG (panel ii), AUG:CAU (panel iii) and UAG:CUA (panel iv) codon:anticodon pairs. Star symbols in the boxes indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in SA (C) for the CAC:GUG, CAU:GUG, AUG:CAU and UAG:CUA codon:anticodon pairs were 1462 ± 374, 493 ± 64, 7314 ± 923 and 5246 ± 763 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Figure 3.

Initiation activities of the native tRNA from various codons. Star symbols in the box indicate G966 (anticodon proximal) and C967. Fold differences in the CAT activities with respect to SA (C, taken as 1) in its ΔrsmB, ΔrsmD, and ΔrsmB ΔrsmD derivatives are shown. For reference, the average CAT activities in the SA control (C) for the AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG constructs were 7314 ± 923, 3629 ± 492, 4019 ± 1000, 906 ± 132, 238 ± 33, 301 ± 32, 352.5 ± 38 and 48 ± 15 pmol Cm acetylated per 1 μg of total cell-free extract in 20 min at 37°C, respectively.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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 tRNA^fMet 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.

Figure 7.

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 tRNA^fMet 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.