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Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation

View Article: PubMed Central - PubMed

ABSTRACT

In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition.

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Related in: MedlinePlus

SD/ASD and PICs 1C and 2A, Related to Figure 5(A) The SD:ASD helix with SD (magenta) and ASD in PIC-1A and PIC-4 (both cyan) and PIC-2B (gray). The SD/ASD in PIC-2B moves ∼11 Å relative to its position in the other PICs. Proteins bS18, uS11 and uS7 are also shown lining the exit channel; The uS7 in PIC-2B (gray) would be too close to the SD/ASD helix in the other PICs.(B) Another view of SD:ASD helices with SD (magenta) and ASD in PIC-1A (cyan) and PIC-2B (gray). A straight mRNA path to the P site is seen for PIC-1A but not for PIC-2B. The uS7 in PIC-2B (gray) would clash with the original position of SD:ASD in PIC-1A. Residue A1503 is also shown away from the mRNA.(C) Interactions of IF3 with the mRNA at the P site in PIC-1C. Arg159 (part of the β-hairpin) is in the vicinity of A+1 and U+2.(D) Top panel: h29 containing G1338 and A1339 (shown as spheres) is positioned optimally in PIC-1C (yellow) similar to that in PIC-2A (cyan) to interact with conserved GCs in ASL of tRNA. However, the h29 (gray) in PIC-1A would clash with ASL. The tRNA from PIC-2A is shown in green surface. Bottom panel: Recognition of conserved GCs in ASL by G1338 and A1339 of h29 in PIC-4.(E) Interactions of IF3 with the mRNA at the P site in PIC-2A. Arg125 of CTD is in the vicinity of the +4 nucleotide of mRNA. The tail of uS9 interacts with G966 and the ASL. The uS13 tail residues also H-bond with the ASL.(F) Comparison of the P site and tRNA and mRNA in PIC-2A (open conformation) and PIC-4 (closed conformation). The 30S, tRNA and mRNA in PIC-2A is shown in color. The tRNA and mRNA in PIC-4 are shown in gray. A widened P site can be seen with the ASL and the codon positioned slightly away from body in the left panel. While in the right panel the 30S head in PIC-4 (in gray) is closer to the body with comparatively narrower P site and the ASL and codon move closer to the body.
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figs5: SD/ASD and PICs 1C and 2A, Related to Figure 5(A) The SD:ASD helix with SD (magenta) and ASD in PIC-1A and PIC-4 (both cyan) and PIC-2B (gray). The SD/ASD in PIC-2B moves ∼11 Å relative to its position in the other PICs. Proteins bS18, uS11 and uS7 are also shown lining the exit channel; The uS7 in PIC-2B (gray) would be too close to the SD/ASD helix in the other PICs.(B) Another view of SD:ASD helices with SD (magenta) and ASD in PIC-1A (cyan) and PIC-2B (gray). A straight mRNA path to the P site is seen for PIC-1A but not for PIC-2B. The uS7 in PIC-2B (gray) would clash with the original position of SD:ASD in PIC-1A. Residue A1503 is also shown away from the mRNA.(C) Interactions of IF3 with the mRNA at the P site in PIC-1C. Arg159 (part of the β-hairpin) is in the vicinity of A+1 and U+2.(D) Top panel: h29 containing G1338 and A1339 (shown as spheres) is positioned optimally in PIC-1C (yellow) similar to that in PIC-2A (cyan) to interact with conserved GCs in ASL of tRNA. However, the h29 (gray) in PIC-1A would clash with ASL. The tRNA from PIC-2A is shown in green surface. Bottom panel: Recognition of conserved GCs in ASL by G1338 and A1339 of h29 in PIC-4.(E) Interactions of IF3 with the mRNA at the P site in PIC-2A. Arg125 of CTD is in the vicinity of the +4 nucleotide of mRNA. The tail of uS9 interacts with G966 and the ASL. The uS13 tail residues also H-bond with the ASL.(F) Comparison of the P site and tRNA and mRNA in PIC-2A (open conformation) and PIC-4 (closed conformation). The 30S, tRNA and mRNA in PIC-2A is shown in color. The tRNA and mRNA in PIC-4 are shown in gray. A widened P site can be seen with the ASL and the codon positioned slightly away from body in the left panel. While in the right panel the 30S head in PIC-4 (in gray) is closer to the body with comparatively narrower P site and the ASL and codon move closer to the body.

Mentions: A double helix formed by base-pairing between the mRNA SD and ASD is observed in all PICs. The mRNA path and the location of the SD:ASD helix seems to be affected by the presence of IFs (IF1+IF3). In the structures reported here, the SD:ASD helix lies closer to uS11 and uS7 (Figure S5A), and farther from uS2, than in other structures lacking IFs (Jenner et al., 2010). This new and putatively IF-dependent SD:ASD position may impart to the mRNA a roughly straight path from the E site to the P site, which could be important to allow sliding of mRNA for positioning the start codon in the P site. This mRNA path is also more distant from A1503 (Figure S5B), which has been proposed based on some crystal structures to function as a pawl to prevent mRNA slippage just after translocation (Zhou et al., 2013). The SD:ASD helix occupies the same position in all PICs except in PIC-2B where it is slightly displaced from uS7 and uS11 (Figures S5A and S5B).


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
SD/ASD and PICs 1C and 2A, Related to Figure 5(A) The SD:ASD helix with SD (magenta) and ASD in PIC-1A and PIC-4 (both cyan) and PIC-2B (gray). The SD/ASD in PIC-2B moves ∼11 Å relative to its position in the other PICs. Proteins bS18, uS11 and uS7 are also shown lining the exit channel; The uS7 in PIC-2B (gray) would be too close to the SD/ASD helix in the other PICs.(B) Another view of SD:ASD helices with SD (magenta) and ASD in PIC-1A (cyan) and PIC-2B (gray). A straight mRNA path to the P site is seen for PIC-1A but not for PIC-2B. The uS7 in PIC-2B (gray) would clash with the original position of SD:ASD in PIC-1A. Residue A1503 is also shown away from the mRNA.(C) Interactions of IF3 with the mRNA at the P site in PIC-1C. Arg159 (part of the β-hairpin) is in the vicinity of A+1 and U+2.(D) Top panel: h29 containing G1338 and A1339 (shown as spheres) is positioned optimally in PIC-1C (yellow) similar to that in PIC-2A (cyan) to interact with conserved GCs in ASL of tRNA. However, the h29 (gray) in PIC-1A would clash with ASL. The tRNA from PIC-2A is shown in green surface. Bottom panel: Recognition of conserved GCs in ASL by G1338 and A1339 of h29 in PIC-4.(E) Interactions of IF3 with the mRNA at the P site in PIC-2A. Arg125 of CTD is in the vicinity of the +4 nucleotide of mRNA. The tail of uS9 interacts with G966 and the ASL. The uS13 tail residues also H-bond with the ASL.(F) Comparison of the P site and tRNA and mRNA in PIC-2A (open conformation) and PIC-4 (closed conformation). The 30S, tRNA and mRNA in PIC-2A is shown in color. The tRNA and mRNA in PIC-4 are shown in gray. A widened P site can be seen with the ASL and the codon positioned slightly away from body in the left panel. While in the right panel the 30S head in PIC-4 (in gray) is closer to the body with comparatively narrower P site and the ASL and codon move closer to the body.
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figs5: SD/ASD and PICs 1C and 2A, Related to Figure 5(A) The SD:ASD helix with SD (magenta) and ASD in PIC-1A and PIC-4 (both cyan) and PIC-2B (gray). The SD/ASD in PIC-2B moves ∼11 Å relative to its position in the other PICs. Proteins bS18, uS11 and uS7 are also shown lining the exit channel; The uS7 in PIC-2B (gray) would be too close to the SD/ASD helix in the other PICs.(B) Another view of SD:ASD helices with SD (magenta) and ASD in PIC-1A (cyan) and PIC-2B (gray). A straight mRNA path to the P site is seen for PIC-1A but not for PIC-2B. The uS7 in PIC-2B (gray) would clash with the original position of SD:ASD in PIC-1A. Residue A1503 is also shown away from the mRNA.(C) Interactions of IF3 with the mRNA at the P site in PIC-1C. Arg159 (part of the β-hairpin) is in the vicinity of A+1 and U+2.(D) Top panel: h29 containing G1338 and A1339 (shown as spheres) is positioned optimally in PIC-1C (yellow) similar to that in PIC-2A (cyan) to interact with conserved GCs in ASL of tRNA. However, the h29 (gray) in PIC-1A would clash with ASL. The tRNA from PIC-2A is shown in green surface. Bottom panel: Recognition of conserved GCs in ASL by G1338 and A1339 of h29 in PIC-4.(E) Interactions of IF3 with the mRNA at the P site in PIC-2A. Arg125 of CTD is in the vicinity of the +4 nucleotide of mRNA. The tail of uS9 interacts with G966 and the ASL. The uS13 tail residues also H-bond with the ASL.(F) Comparison of the P site and tRNA and mRNA in PIC-2A (open conformation) and PIC-4 (closed conformation). The 30S, tRNA and mRNA in PIC-2A is shown in color. The tRNA and mRNA in PIC-4 are shown in gray. A widened P site can be seen with the ASL and the codon positioned slightly away from body in the left panel. While in the right panel the 30S head in PIC-4 (in gray) is closer to the body with comparatively narrower P site and the ASL and codon move closer to the body.
Mentions: A double helix formed by base-pairing between the mRNA SD and ASD is observed in all PICs. The mRNA path and the location of the SD:ASD helix seems to be affected by the presence of IFs (IF1+IF3). In the structures reported here, the SD:ASD helix lies closer to uS11 and uS7 (Figure S5A), and farther from uS2, than in other structures lacking IFs (Jenner et al., 2010). This new and putatively IF-dependent SD:ASD position may impart to the mRNA a roughly straight path from the E site to the P site, which could be important to allow sliding of mRNA for positioning the start codon in the P site. This mRNA path is also more distant from A1503 (Figure S5B), which has been proposed based on some crystal structures to function as a pawl to prevent mRNA slippage just after translocation (Zhou et al., 2013). The SD:ASD helix occupies the same position in all PICs except in PIC-2B where it is slightly displaced from uS7 and uS11 (Figures S5A and S5B).

View Article: PubMed Central - PubMed

ABSTRACT

In bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition.

No MeSH data available.


Related in: MedlinePlus