The focus of this proposal is the mechanism of mammalian translation initiation, which requires at least 9 initiation factors (eIFs), and is a target for multiple regulatory pathways. It occurs in two stages: formation of a 48S initiation complex at the initiation codon of mRNA and its joining with a 60S ribosomal subunit. First, 43S preinitiation complex comprising a 40S ribosomal subunit, a ternary complex of eIF2, initiator tRNA and GTP, and eIFs 3, 1 and 1A attaches to the capped 5'-proximal region of mRNA in a step that involves unwinding of its secondary structure by eIFs 4A, 4B and 4F, and then scans to the initiation codon. After initiation codon recognition and formation of the 48S complex with established codon-anticodon base-pairing, eIF5 and eIF5B promote hydrolysis of eIF2-bound GTP, displacement of eIFs from the 40S subunit and joining of a 60S subunit. The proposed studies will be based on the approach of in vitro reconstitution of all stages of protein synthesis (initiation, elongation, termination and ribosomal recycling) from individual purified translational components.
In Aim 1, we will investigate the mechanistic aspects of entry of eIF4F-bound capped mRNAs into the mRNA-binding cleft of the 40S subunit during attachment of 43S complexes by determining the position of eIF4E in ribosomal initiation complexes, identifying the first position in a capped mRNA at which an AUG codon can interact productively with initiator tRNA, and following the fate of the cap-eIF4E- eIF4G-eIF3-40S chain of interactions during the transition from ribosomal attachment to scanning.
In Aim 2, we propose to investigate the network of DEAD/DExH-box proteins that have currently been implicated in initiation (e.g. eIF4A, Ded1, DHX29 etc.) by characterizing their relative individual activities at distinct stages of initiation (ribosomal attachment and scanning) and their potential synergy in promoting ribosomal scanning through stable mRNA secondary structures. We also propose to develop fast kinetics techniques to measure kinetic parameters of scanning and to determine how they differ depending on the helicases and other factors involved.
Aim 3 will be devoted to investigation of the mechanism of action of various physiologically important translation regulators that have been implicated in protein synthesis by studies in vivo.
In Aim 4, we will characterize mechanisms of post-recycling regulation of initiation, focusing on two processes, preferential shunting of recycled 40S subunits back to the 5'-end of the same mRNA, and reinitiation after translation of short open reading frames.
Protein synthesis is of central importance in cell metabolism, and its complex initiation stage is a target for multiple regulatory pathways that integrate it with developmental processes and with changes in the cellular environment. Accordingly, defects in the initiation process can cause severe inherited diseases such as hereditary thrombocythemia and congenital erythroid aplasia, and aberrant cell growth and proliferation, for example in tumors. These studies will determine the molecular basis for key events in translation initiation, and its regulation by trans-acting factors, which is a prerequisite for the development of rational therapies to treat such diseases. )
|Kumar, Parimal; Sweeney, Trevor R; Skabkin, Maxim A et al. (2014) Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs. Nucleic Acids Res 42:3228-45|
|Hashem, Yaser; des Georges, Amedee; Dhote, Vidya et al. (2013) Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell 153:1108-19|
|Hashem, Yaser; des Georges, Amedee; Dhote, Vidya et al. (2013) Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature 503:539-43|
|Skabkin, Maxim A; Skabkina, Olga V; Hellen, Christopher U T et al. (2013) Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol Cell 51:249-64|
|Abaeva, Irina S; Marintchev, Assen; Pisareva, Vera P et al. (2011) Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning. EMBO J 30:115-29|
|Yu, Yingpu; Abaeva, Irina S; Marintchev, Assen et al. (2011) Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors. Nucleic Acids Res 39:4851-65|
|Yu, Yingpu; Sweeney, Trevor R; Kafasla, Panagiota et al. (2011) The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES. EMBO J 30:4423-36|
|Skabkin, Maxim A; Skabkina, Olga V; Dhote, Vidya et al. (2010) Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev 24:1787-801|
|Jackson, Richard J; Hellen, Christopher U T; Pestova, Tatyana V (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113-27|
|Yu, Yingpu; Marintchev, Assen; Kolupaeva, Victoria G et al. (2009) Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing. Nucleic Acids Res 37:5167-82|
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