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

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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.


Schematic of Major Conformational Changes during InitiationThe various PIC structures are summarized in an order that represents one possible initiation pathway in which mRNA binding precedes tRNA binding. In such an “mRNA-first” pathway, the 30S head may swivel back and forth (shown by curved arrows) and also move up and down (shown by black arrows). The bold arrows show the direction of the pathway. Solid arrows highlight one pathway, while the dashed arrows show possible alternative pathways. The movement of CTD, NTD, and tRNA is shown schematically.See also Movie S5.
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fig7: Schematic of Major Conformational Changes during InitiationThe various PIC structures are summarized in an order that represents one possible initiation pathway in which mRNA binding precedes tRNA binding. In such an “mRNA-first” pathway, the 30S head may swivel back and forth (shown by curved arrows) and also move up and down (shown by black arrows). The bold arrows show the direction of the pathway. Solid arrows highlight one pathway, while the dashed arrows show possible alternative pathways. The movement of CTD, NTD, and tRNA is shown schematically.See also Movie S5.

Mentions: All three bacterial IFs cooperate to make translation initiation faster and more accurate. However, a large body of genetic, biochemical, and biophysical data indicate that IF3 plays a particularly important and dynamic role in maintaining the fidelity of initiation. Classic in vitro toeprinting experiments showed that IF3 is necessary and sufficient to discriminate against elongator tRNAs (Hartz et al., 1989) as well as many non-cognate start codons (Hartz et al., 1990). The eight IF2-free structures (PICs 1–4) presented here provide snapshots of different states of the bacterial 30S preinitiation complex with IF3 in different conformational states. Although at lower resolution, the structures of the complete 30S initiation complex (PICs I–III) with all three IFs, fMet-tRNAfMet, and mRNA show that the conformations and positions of the tRNA and factors are very similar to those seen in the IF2-free structures, thus validating the conclusions drawn from those higher-resolution structures. Altogether these structures provide many new insights into key stages of translation initiation (Figure 7; Movie S5), and the various IF3 states suggest possible mechanisms for its roles in the translation initiation pathway.


Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation
Schematic of Major Conformational Changes during InitiationThe various PIC structures are summarized in an order that represents one possible initiation pathway in which mRNA binding precedes tRNA binding. In such an “mRNA-first” pathway, the 30S head may swivel back and forth (shown by curved arrows) and also move up and down (shown by black arrows). The bold arrows show the direction of the pathway. Solid arrows highlight one pathway, while the dashed arrows show possible alternative pathways. The movement of CTD, NTD, and tRNA is shown schematically.See also Movie S5.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig7: Schematic of Major Conformational Changes during InitiationThe various PIC structures are summarized in an order that represents one possible initiation pathway in which mRNA binding precedes tRNA binding. In such an “mRNA-first” pathway, the 30S head may swivel back and forth (shown by curved arrows) and also move up and down (shown by black arrows). The bold arrows show the direction of the pathway. Solid arrows highlight one pathway, while the dashed arrows show possible alternative pathways. The movement of CTD, NTD, and tRNA is shown schematically.See also Movie S5.
Mentions: All three bacterial IFs cooperate to make translation initiation faster and more accurate. However, a large body of genetic, biochemical, and biophysical data indicate that IF3 plays a particularly important and dynamic role in maintaining the fidelity of initiation. Classic in vitro toeprinting experiments showed that IF3 is necessary and sufficient to discriminate against elongator tRNAs (Hartz et al., 1989) as well as many non-cognate start codons (Hartz et al., 1990). The eight IF2-free structures (PICs 1–4) presented here provide snapshots of different states of the bacterial 30S preinitiation complex with IF3 in different conformational states. Although at lower resolution, the structures of the complete 30S initiation complex (PICs I–III) with all three IFs, fMet-tRNAfMet, and mRNA show that the conformations and positions of the tRNA and factors are very similar to those seen in the IF2-free structures, thus validating the conclusions drawn from those higher-resolution structures. Altogether these structures provide many new insights into key stages of translation initiation (Figure 7; Movie S5), and the various IF3 states suggest possible mechanisms for its roles in the translation initiation pathway.

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.