In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNA i in a "P IN " state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNA i . Substitutions of eIF5 residues involved in the eIF5-NTD/tRNA i interaction influenced initiation at near-cognate UUG codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection. IntroductionEukaryotic translation initiation is a multistep process that involves assembly of a preinitiation complex (PIC) comprised of the small (40S) ribosomal subunit, methionyl initiator tRNA (Met-tRNA i ) and numerous eukaryotic initiation factors (eIFs). The binding of this 43S PIC to the capped 5ʹ end of mRNA is followed by scanning the mRNA leader for the correct AUG start codon. The binding of eIF1 and eIF1A to the 40S subunit promotes a scanningconducive, open conformation favourable for rapid binding of Met-tRNA i as a ternary complex (TC) with eIF2-GTP, in a conformation (P OUT ) suitable for scanning successive triplets in the 40S P site for complementarity to the anticodon of Met-tRNA i . The multisubunit eIF3 complex also binds directly to the 40S subunit and stimulates 43S assembly, attachment to mRNA, and subsequent scanning. During the scanning process, 3 hydrolysis of GTP in TC is stimulated by the GTPase activating protein (GAP) eIF5, but release of phosphate (P i ) from eIF2-GDP-P i is prevented by the gatekeeper molecule eIF1 at non-AUG codons. Recognition of an AUG start codon induces a major conformational change in the PIC to a scanning-arrested closed (P IN ) complex made possible by the dissociation of eIF1, which eliminates a clash it would have with Met-tRNA i in its fully accommodated P IN conformation. The change is accompanied by the movement of the Cterminal tail (CTT) of eIF1A from eIF1 towards the GAP domain of eIF5, an event that eIF1 binds in the open/P IN state. The tRNA i is more fully accommodated in the P site than observed in previous structures containing eIF1, and is also tilted toward the 40S body, apparently setting the stage for its interaction with eIF5B and subsequent joining of the 60S subunit. Extensive interaction with the eIF5-NTD appears to stabilize this tRNA i conformation, using two β-hairpins structurally analogous to those in eIF1 that oppose tRNA i accommodation in the scanning complex. Mutations expected to weaken the observed eIF5-NTD/tRNA i interactions diminish ...

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3 H-labeled and 14 C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

Breast cancer, also recognized as the principal cause of cancer-related deaths among women, is the second most

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