Initiation of translation in the majority of eukaryotic cellular mRNAs depends on the 5'-cap (m7GpppN) and involves ribosomal scanning of the 5' untranslated region (5'-UTR) for an initiating AUG start codon. Therefore, this mechanism is often called cap-dependent translation initiation. Proximity to the cap, as well as the nucleotides surrounding an AUG codon, influence the efficiency of the start site recognition during the scanning process. However, if the recognition site is poor enough, scanning ribosomal subunits will ignore and skip potential starting AUGs, a phenomenon called leaky scanning. Leaky scanning allows a single mRNA to encode several proteins that differ in their amino-termini. Merrick (2010) provides an overview of this process and hghlights several features of it that remain incompletely understood.
Several eukaryotic cell and viral mRNAs initiate translation by an alternative mechanism that involves internal initiation rather than ribosomal scanning. These mRNAs contain complex nucleotide sequences, called internal ribosomal entry sites, where ribosomes bind in a cap-independent manner and start translation at the closest downstream AUG codon.
Initiation on several viral and cellular mRNAs is cap-independent and is mediated by binding of the ribosome to internal ribosome entry site (IRES) elements. These elements are often found in characteristically long structured regions on the 5'-UTR of an mRNA that may or may not have regulatory upstream open reading frames (uORFs). Both of these features on the 5'-end of the mRNA hinder ribosomal scanning, and thus promote a cap-independent translation initiation mechanism. IRESs act as specific translational enhancers that allow translation initiation to occur in response to specific stimuli and under the control of different trans-acting factors, as for example when cap-dependent protein synthesis is shut off during viral infection. Such regulatory elements have been identified in the mRNAs of growth factors, protooncogenes, angiogenesis factors, and apoptosis regulators, which are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and apoptosis. Thus, cap-independent translational control might have evolved to regulate cellular responses in acute but transient stress conditions that would otherwise lead to cell death, while the same mechanism is of major importance for viral mRNAs to bypass the shutting-off of host protein synthesis after infection. Encephalomyocarditis virus (EMCV) and hepatitis C virus exemplify two distinct mechanisms of IRES-mediated initiation. In contrast to cap-dependent initiation, the eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of a 43S complex. Accordingly, EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Nonetheless, initiation on some EMCV-like IRESs requires additional non-canonical initiation factors, which alter IRES conformation and promote binding of eIF4A/eIF4G. Initiation on the hepatitis C virus IRES is simpler: a 43S complex containing only eIF2 and eIF3 binds directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.
Pause A, Belsham GJ, Gingras AC, Donzé O, Lin TA, Lawrence JC, Sonenberg N.; ''Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function.''; PubMedEurope PMCScholia
Sampath P, Mazumder B, Seshadri V, Fox PL.; ''Transcript-selective translational silencing by gamma interferon is directed by a novel structural element in the ceruloplasmin mRNA 3' untranslated region.''; PubMedEurope PMCScholia
Pestova TV, Borukhov SI, Hellen CU.; ''Eukaryotic ribosomes require initiation factors 1 and 1A to locate initiation codons.''; PubMedEurope PMCScholia
Mazumder B, Sampath P, Seshadri V, Maitra RK, DiCorleto PE, Fox PL.; ''Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control.''; PubMedEurope PMCScholia
Safer B, Adams SL, Anderson WF, Merrick WC.; ''Binding of MET-TRNAf and GTP to homogeneous initiation factor MP.''; PubMedEurope PMCScholia
Pestova TV, Shatsky IN, Hellen CU.; ''Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.''; PubMedEurope PMCScholia
Kozak M.; ''Evaluation of the "scanning model" for initiation of protein synthesis in eucaryotes.''; PubMedEurope PMCScholia
Chakrabarti A, Maitra U.; ''Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.''; PubMedEurope PMCScholia
Trachsel H, Erni B, Schreier MH, Staehelin T.; ''Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors.''; PubMedEurope PMCScholia
Rowlands AG, Panniers R, Henshaw EC.; ''The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2.''; PubMedEurope PMCScholia
Iost I, Dreyfus M, Linder P.; ''Ded1p, a DEAD-box protein required for translation initiation in Saccharomyces cerevisiae, is an RNA helicase.''; PubMedEurope PMCScholia
Benne R, Hershey JW.; ''The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes.''; PubMedEurope PMCScholia
Schreier MH, Erni B, Staehelin T.; ''Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors.''; PubMedEurope PMCScholia
Sonenberg N, Rupprecht KM, Hecht SM, Shatkin AJ.; ''Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.''; PubMedEurope PMCScholia
Dever TE, Wei CL, Benkowski LA, Browning K, Merrick WC, Hershey JW.; ''Determination of the amino acid sequence of rabbit, human, and wheat germ protein synthesis factor eIF-4C by cloning and chemical sequencing.''; PubMedEurope PMCScholia
Merrick WC, Kemper WM, Anderson WF.; ''Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.''; PubMedEurope PMCScholia
Asano K, Clayton J, Shalev A, Hinnebusch AG.; ''A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNA(Met) is an important translation initiation intermediate in vivo.''; PubMedEurope PMCScholia
Peterson DT, Merrick WC, Safer B.; ''Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.''; PubMedEurope PMCScholia
Goumans H, Thomas A, Verhoeven A, Voorma HO, Benne R.; ''The role of eIF-4C in protein synthesis initiation complex formation.''; PubMedEurope PMCScholia
Imataka H, Gradi A, Sonenberg N.; ''A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation.''; PubMedEurope PMCScholia
Chuang RY, Weaver PL, Liu Z, Chang TH.; ''Requirement of the DEAD-Box protein ded1p for messenger RNA translation.''; PubMedEurope PMCScholia
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The precise order of events leading to the circularization of poly (A) mRNA during translation initiation is unknown. Here the association of PABP with the poly (A) mRNA and the association of PABP with eIF4F are represented as occuring simultaneously after formation of the initiation complex. However, it is also possible that these interactions occur during the formation of the translation initiation complex. The binding of eIF4F to the cap and binding of PABP to the poly (A) tail, for example, may occur at the same time. In fact, the eIF4G-PABP interaction helps eIF4F to bind tighter to the cap (Borman et al. 2000.) In addition, eIF4B and eIF4H bind more transiently to the mRNA and may not be part of an initial complex in which PABP has not yet touched eIF4G.
Although the mechanism through which L13a prevents translation initiation has not been determined, Mazumder et al. (2003) have described four alternatives. L13a could (1) inhibit the function of eIF4F, (2) block the recruitment of the 43S preinitiation complex, (3) prevent scanning of the 43S complex to the initiation codon, or 4) interfere with joining of the 60S ribosomal subunit.
The L13a subunit of the 60s ribosome is phosphorylated about 16 hours after INF gamma induction by an unknown kinase. At this time, L13a is also released from the 60s subunit (Mazumder et al.,2003). It is unclear, however, whether phosphorylation occurs before or after the release of L13a. Here, phosphorylation is shown as occurring after release.
The L13a subunit of the 60s ribosome is phosphorylated about 16 hours after INF gamma induction by an unknown kinase. At this time, L13a is also released from the 60s subunit (Mazumder et al.,2003). It is unclear, however, whether phosphorylation occurs before or after the release of L13a. Here, phosphorylation is shown as occurring after release.
The translation initiation complex forms when the 43S complex binds the mRNA that is associated with eIF4F, eIF4B and eIF4H. eIF4G in the eIF4F complex can directly contact eIF3 in the 43S complex. eIF1A is necessary for the formation of this complex.
Once the Met-tRNAi has recognized the AUG, eIF2-bound GTP is hydrolyzed. The reaction is catalyzed by eIF5 (or eIF5B) and is thought to cause dissociation of all other initiation factors and allow joining of the large 60S ribosomal subunit. Release of the initiation factors from 40S leaves the Met-tRNAi in the ribosomal P-site base-paired to the start codon on the mRNA.
The mRNA-bound ribosomal complex moves along the 5'-untranslated region (5'-UTR) of the mRNA from its initial site to the initiation codon to form a 48S complex, in which the initiation codon (AUG) is base paired to the anticodon of the Met-tRNAi. It is not known whether eIF4A (or another ATPase, such as DED1) facilitates scanning by melting mRNA secondary structures or by actively propelling the ribosome.
The factor eIF4E within the eIF4F (cap-binding) complex directly binds the 5'-cap on eukaryotic mRNAs. Note that the mRNA is in complex with cytoplasmic proteins constituting an mRNP complex.
The DEAD-box RNA helicase eIF4A, together with the RNA-binding proteins eIF4B or eIF4H, is thought to unwind RNA secondary structures near the 5'-end of the mRNA and in the presence of ATP.
Once the 60S subunit joins the translation initiation complex, eIF5B hydrolyzes its GTP and is released from the now 80S monosome. The fully assembled 80s ribosome is now ready to start elongation of the polypeptide chain.
The ternary complex (Met-tRNAi:eIF2:GTP) binds to the complex formed by the 40S subunit, eIF3 and eIF1A, to form the 43S complex. eIF1A promotes binding of the ternary complex to the 40S subunit within 43S. The initiator methionyl-tRNA from the ternary complex is positioned at the ribosomal P site.
The AUG initiation codon in the mRNA is recognized by base pairing with the anticodon of the Met-tRNAi. This reaction requires eIF1, eIF1A, eIF2 and eIF5.
eIF2B is a guanine nucleotide releasing factor that is required to cause GDP release so that a new GTP molecule can bind and activate eIF2, so that it can be reused.
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Ceruloplasmin
mRNA:eIF4F:eIF4B:eIF4H:PABPcomplex lacking
L13a subunitin RNP (ribonucleoprotein)
complexesassociated wth the 3' UTR GAIT element of ceruloplasmin mRNA within the translation
initiation complexAnnotated Interactions
Ceruloplasmin
mRNA:eIF4F:eIF4B:eIF4H:PABPCeruloplasmin
mRNA:eIF4F:eIF4B:eIF4H:PABPcomplex lacking
L13a subunitin RNP (ribonucleoprotein)
complexesassociated wth the 3' UTR GAIT element of ceruloplasmin mRNA within the translation
initiation complex