Limits...
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.

No MeSH data available.


Contacts of IF3 at the P Site(A) In PIC-1C, the A+1 base stacks with A790, while the −1 base stacks with G926. C1400 and U1498 are also shown. Residues of IF3 close to the codon are shown.(B) In the presence of the fMet-tRNAfMet in PIC-2A, the A+1 and U+2 flip to interact with the anticodon.(C) In PIC-2C, the CTD remains in position 1 although no longer close to the codon as seen in PIC-2A.(D) Codon:anticodon interaction in PIC-3 as the CTD is adjusted to position 1′. The Arg123 and Arg159 of IF3 lie close to ASL at the P site.(E) Codon:anticodon interaction in PIC-4 as the CTD is moved away. Arg128 of uS9 interacts with ASL.(F) Similar orientation of the P site in PIC-2B is shown in earlier complexes. The codon:anticodon is moved toward E (dashed arrow) site such that G+3 now occupies the position of A+1. mRNA from PIC-4 is shown in gray.See also Figures S5 and S6 and Movie S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037330&req=5

fig5: Contacts of IF3 at the P Site(A) In PIC-1C, the A+1 base stacks with A790, while the −1 base stacks with G926. C1400 and U1498 are also shown. Residues of IF3 close to the codon are shown.(B) In the presence of the fMet-tRNAfMet in PIC-2A, the A+1 and U+2 flip to interact with the anticodon.(C) In PIC-2C, the CTD remains in position 1 although no longer close to the codon as seen in PIC-2A.(D) Codon:anticodon interaction in PIC-3 as the CTD is adjusted to position 1′. The Arg123 and Arg159 of IF3 lie close to ASL at the P site.(E) Codon:anticodon interaction in PIC-4 as the CTD is moved away. Arg128 of uS9 interacts with ASL.(F) Similar orientation of the P site in PIC-2B is shown in earlier complexes. The codon:anticodon is moved toward E (dashed arrow) site such that G+3 now occupies the position of A+1. mRNA from PIC-4 is shown in gray.See also Figures S5 and S6 and Movie S4.

Mentions: In tRNA-free PICs 1A–1C, we observe an unusual mRNA conformation, in which a base at −1 position of mRNA stacks with G926 in the E site, while the next base, the A+1 of the AUG codon, stacks with A790 in the P site (Figure 5A). The base stacking by the −1 base of mRNA is also observed in the case of eukaryotic complexes. The A+1 is flipped toward the 30S body and makes an H-bond with Arg91 of CTD (Figures 5A and S5C). The U+2 of AUG is also flipped and its backbone interacts with Arg123 of CTD. Arg159 of CTD is in the vicinity of A+1 and U+2. The G+3 is not flipped and may interact with C1400. G+3 is in a state ready to base pair with the anticodon, while the +4 nucleotide backbone makes an H-bond with Gly124 of CTD (Figure S5C).


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Contacts of IF3 at the P Site(A) In PIC-1C, the A+1 base stacks with A790, while the −1 base stacks with G926. C1400 and U1498 are also shown. Residues of IF3 close to the codon are shown.(B) In the presence of the fMet-tRNAfMet in PIC-2A, the A+1 and U+2 flip to interact with the anticodon.(C) In PIC-2C, the CTD remains in position 1 although no longer close to the codon as seen in PIC-2A.(D) Codon:anticodon interaction in PIC-3 as the CTD is adjusted to position 1′. The Arg123 and Arg159 of IF3 lie close to ASL at the P site.(E) Codon:anticodon interaction in PIC-4 as the CTD is moved away. Arg128 of uS9 interacts with ASL.(F) Similar orientation of the P site in PIC-2B is shown in earlier complexes. The codon:anticodon is moved toward E (dashed arrow) site such that G+3 now occupies the position of A+1. mRNA from PIC-4 is shown in gray.See also Figures S5 and S6 and Movie S4.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037330&req=5

fig5: Contacts of IF3 at the P Site(A) In PIC-1C, the A+1 base stacks with A790, while the −1 base stacks with G926. C1400 and U1498 are also shown. Residues of IF3 close to the codon are shown.(B) In the presence of the fMet-tRNAfMet in PIC-2A, the A+1 and U+2 flip to interact with the anticodon.(C) In PIC-2C, the CTD remains in position 1 although no longer close to the codon as seen in PIC-2A.(D) Codon:anticodon interaction in PIC-3 as the CTD is adjusted to position 1′. The Arg123 and Arg159 of IF3 lie close to ASL at the P site.(E) Codon:anticodon interaction in PIC-4 as the CTD is moved away. Arg128 of uS9 interacts with ASL.(F) Similar orientation of the P site in PIC-2B is shown in earlier complexes. The codon:anticodon is moved toward E (dashed arrow) site such that G+3 now occupies the position of A+1. mRNA from PIC-4 is shown in gray.See also Figures S5 and S6 and Movie S4.
Mentions: In tRNA-free PICs 1A–1C, we observe an unusual mRNA conformation, in which a base at −1 position of mRNA stacks with G926 in the E site, while the next base, the A+1 of the AUG codon, stacks with A790 in the P site (Figure 5A). The base stacking by the −1 base of mRNA is also observed in the case of eukaryotic complexes. The A+1 is flipped toward the 30S body and makes an H-bond with Arg91 of CTD (Figures 5A and S5C). The U+2 of AUG is also flipped and its backbone interacts with Arg123 of CTD. Arg159 of CTD is in the vicinity of A+1 and U+2. The G+3 is not flipped and may interact with C1400. G+3 is in a state ready to base pair with the anticodon, while the +4 nucleotide backbone makes an H-bond with Gly124 of CTD (Figure S5C).

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.