Study with Quizlet and memorize flashcards containing terms like _____ is the first protein to bind to mRNA to being initiation of translation. A. TFIIA B. eIF-2 C. TFIID D. 60S ribosomal subunit E. 40S ribosomal subunit, What part of the ribosome is important in catalyzing peptide bonds? A. the E site B. the methyl guanosine cap C. the rRNA D. the ribosomal proteins, Overexpression of miRNAs ...
During the Translation process, eIF-2 initiation factor binds to GTP which then binds to methionine initiator tRNA to form a 43S pre-initiation complex that helps in the formation of 40S ribosomal complex ti initiate translation in the cytoplasm of the cell.
Start studying Chapter 16 Study Questions. Learn vocabulary, terms, and more with flashcards, games, and other study tools.
eIF-2 binds GTP, then eIF-2/GTP binds Met-tRNA, then eIF-1 and eIF-2 bind small ribosomal subunit, then eIF-4 guides the the complexes together to the 5' end of mRNA, then the large ribosomal subunit binds
a lipid of the ER membrane that acts as a carrier for the universal sugar as it is being built
stop codon enters A-site, then eRF bound to GTP binds with codon, then the protein chain is released , then GTP is hydrolyzed , then the transcription complex disassociates
adds a phosphate to a hydroxyl group to activate or inactivate the protein
aminoacyl tRNA binds at A-site, then peptidyltransferase of the large ribosomal subunit forms a peptide bond between the adjacent amino acids, then the tRNA translocates to the P-site, then to the E-site
a leader sequence on the N-terminal is synthesized as the protein is being synthesized, then guided into the mitochondria via the TOM/TIM complex, then the leader sequence is cleaved off
postranslational modification of mannose-6-phosphate targets the enzyme to the lysosome
eIF2 activity is regulated by a mechanism involving both guanine nucleotide exchange and phosphorylation. Phosphorylation takes place at the α-subunit, which is a target for a number of serine kinases that phosphorylate serine 51. Those kinases act as a result of stress such as amino acid deprivation ( GCN2 ), ER stress ( PERK ), the presence of dsRNA ( PKR) heme deficiency ( HRI ), or interferon. Once phosphorylated, eIF2 shows increased affinity for eIF2B, its GEF. However, eIF2B is able to exchange GDP for GTP only if eIF2 is in its unphosphorylated state. Phosphorylated eIF2, however, due to its stronger binding, acts as an inhibitor of its own GEF (eIF2B). Since the cellular concentration of eIF2B is much lower than that of eIF2, even a small amount of phosphorylated eIF2 can completely abolish eIF2B activity by sequestration. Without the GEF, eIF2 can no longer be returned to its active (GTP-bound) state. As a consequence, translation comes to a halt since initiation is no longer possible without any available ternary complex. Furthermore, low concentration of ternary complex allows the expression of GCN4 (starved condition), which, in turn, results in increased activation of amino acid synthesis genes
eIF2. Eukaryotic Initiation Factor 2 ( eIF2) is a eukaryotic initiation factor. It is required for most forms of eukaryotic translation initiation. eIF2 mediates the binding of tRNA iMet to the ribosome in a GTP -dependent manner. eIF2 is a heterotrimer consisting of an alpha (also called subunit 1, EIF2S1), a beta (subunit 2, EIF2S2), ...
eIF2 is an essential factor for protein synthesis that forms a ternary complex (TC) with GTP and the initiator Met - tRNA iMet. After its formation, the TC binds the 40S ribosomal subunit to form the 43S preinitiation complex (43S PIC). 43S PIC assembly is believed to be stimulated by the initiation factors eIF1, eIF1A, and the eIF3 complex according to in vitro experiments. The 43S PIC then binds mRNA that has previously been unwound by the eIF4F complex. The 43S PIC and the eIF4F proteins form a new 48S complex on the mRNA, which starts searching along the mRNA for the start codon (AUG). Upon base pairing of the AUG-codon with the Met-tRNA, eIF5 (which is a GTPase-activating protein , or GAP) is recruited to the complex and induces eIF2 to hydrolyse its GTP. This causes eIF2-GDP to be released from this 48S complex and translation begins after recruitment of the 60S ribosomal sub-unit and formation of the 80S initiation complex. Finally, with the help of the guanine nucleotide exchange factor (GEF) eIF2B, the GDP in eIF2 is exchanged for a GTP and the ternary complex reforms for a new round of translation initiation.
The α-subunit contains the main target for phosphorylation, a serine at position 51. It also contains a S1 motif domain, which is a potential RNA binding-site. Therefore, the α-subunit can be considered the regulatory subunit of the trimer.
The γ-subunit comprises three guanine nucleotide-binding sites and is known to be the main docking site for GTP/GDP. It also contains a tRNA-binding cavity that has been shown by X-ray crystallography. A zinc knuckle motif is able to bind one Zn 2+ cation. It is related to some elongation factors like EF-Tu.
Once phosphorylated, eIF2 shows increased affinity for eIF2B, its GEF. However, eIF2B is able to exchange GDP for GTP only if eIF2 is in its unphosphorylated state. Phosphorylated eIF2, however, due to its stronger binding, acts as an inhibitor of its own GEF (eIF2B).
As a consequence, translation comes to a halt since initiation is no longer possible without any available ternary complex. Furthermore, low concentration of ternary complex allows the expression of GCN4 (starved condition), which, in turn, results in increased activation of amino acid synthesis genes.
Translation initiation is a key step for regulating the synthesis of several proteins. In bacteria, translation initiation involves the interaction of the mRNA with the ribosomal small subunit. Additionally, translation initiation factors 1, 2, and 3, and the initiator tRNA, also assemble on the ribosomal small subunit and are essential ...
eIF2-Met-tRNAi-GTP binds the 40S subunit as a ternary complex. The GDP bound form of eIF2 generated by each initiation cycle cannot bind Met-tRNAi and requires exchange of GDP for GTP by the guanine nucleotide exchange factor, eIF2B. eIF2B is inactivated via phosphorylation by GSK3 at a conserved serine residue, S540. Insulin signaling regulates this first step of translation initiation by controlling the phosphorylation state of eIF2B. Insulin induced activation of PKB/Akt causes phosphorylation and inactivation of GSK3 leading to eIF2B dephosphorylation and consequent activation [20]. In parallel, different forms of cellular stress lead to phosphorylation of the eIF2 α subunit, which converts GDP bound eIF2 into an inhibitor of eIF2B. Thus, phosphorylation of eIF2 constitutes a major means of regulating translation [21]. Although the mechanism is not known, insulin treatment leads to dephosphorylation of the α subunit of eIF2 to promote translation [22,23].
The scaffolding protein eIF4G acts to bring together the components of the 4F initiation complex and also directly interacts with eIF3 of the 43S pre-initiation complex. Insulin induces the phosphorylation of eIF4G in an mTOR Complex1 dependent manner, but the kinase that mediates this phosphorylation event and the functional significance of this event is not known [31]. eIF4A is an mRNA helicase that acts in unison with eIF4B and mRNA binding protein to unwind the 5’ secondary structure of mRNA. Upon insulin treatment, S6K1, a critical downstream effector of mTOR Complex1, phosphorylates eIF4B at S422, stimulating its recruitment to the pre-initiation complex [32]. In addition, S6K1 phosphorylates the programmed cell death protein 4 (PDCD4), a tumor suppressor gene at S67, leading to its degradation [33]. PDCD4 binds to eIF4A and is thought to prevent translation by competing with eIF4G for binding to eIF4A or by inhibiting eIF4A helicase activity [33].
Eukaryotic translation initiation is an extremely complex process that requires at least 12 initiation factors (versus three factors in bacteria) to position an initiator methionyl-tRNAiMet in the P-site of the ribosome, base-paired to the correct AUG codon of the mRNA to be translated. Decades of work have elucidated many details of this process, leading to the current model of eukaryotic initiation ( Fig. 6.1; reviewed in Kapp and Lorsch, 2004b; Pestova et al., 2001, 2007 ). Briefly, eukaryotic initiation factor (eIF) 2 forms a ternary complex (TC) with GTP and methionyl-tRNA iMet that brings the methionyl-tRNA iMet onto the 40S ribosomal subunit with the help of eIF1, eIF1A, and eIF3. The resulting 43S complex is thought to bind to the 5′-end of an mRNA, near the 7-methylguanosine cap, and scan in the 3′ direction in search of the AUG start codon. eIF2, with the aid of the GTPase-activating protein eIF5, is able to partially hydrolyze GTP to GDP ⋅ P i prior to start codon recognition, but is unable to release the bound P i. Recognition of the start codon causes a conformational change in the pre-initiation complex that results in release of eIF1 from its binding site on the 40S subunit. Release of eIF1 triggers rapid P i release from eIF2 ⋅ GDP ⋅ P i, making GTP hydrolysis irreversible and allowing downstream events in the pathway to take place. Recognition of the start codon is also thought to result in an additional conformational change that prevents further scanning of the mRNA. A second GTPase, eIF5B, promotes 60S ribosomal subunit joining to the 40S ⋅ mRNA ⋅ methionyl-tRNA iMet complex. GTP hydrolysis by eIF5B reduces the affinity of the factor for the 80S initiation complex and dissociation of eIF5B results in a translationally competent 80S ribosome.
Translation initiation starts with the formation of the 43S preinitiation complex (PIC) consisting of several soluble factors, including the ternary complex (TC; elF2-GTP-Met-tRNAiMet ), which associate with the small ribosomal subunit. In the next step, mRNA is recruited to form the 48S PIC and the entire machinery starts scanning the 5′ untranslated region of the mRNA until the AUG start codon is encountered. The most widely used method to separate 40S and 60S ribosomal subunits from soluble factors, monosomes and polysomes, is sucrose density centrifugation (SDC). Since PICs are intrinsically unstable complexes that cannot withstand the forces imposed by SDC, a stabilization agent must be employed to detect the association of factors with the 40S subunit after SDC. This was initially achieved by adding heparin (a highly sulfated glycosaminoglycan) directly to the breaking buffer of cells treated with cycloheximide (a translation elongation inhibitor). However, the mechanism of stabilization is not understood and, moreover, there are indications that the use of heparin may lead to artifactual factor associations that do not reflect the factor occupancy of the 43S/48S PICs in the cell at the time of lysis. Therefore, we developed an alternative method for PIC stabilization using formaldehyde (HCHO) to cross-link factors associated with 40S ribosomal subunits in vivo before the disruption of the yeast cells. Results obtained using HCHO stabilization strongly indicate that the factors detected on the 43S/48S PIC after SDC approximate a real-time in vivo “snapshot” of the 43S/48S PIC composition. In this chapter, we will present the protocol for HCHO cross-linking in detail and demonstrate the difference between heparin and HCHO stabilization procedures. In addition, different conditions for displaying the polysome profile or PIC analysis by SDC, used to address different questions, will be outlined.
There are two types of mRNA translation initiation: the cap-dependent translation initiation and the cap-independent translation initiation. Furthermore, there is another mRNA category (10%) that is translated in a cap- and eIF4E-independent manner. These mRNAs have a structure called “IRES” (internal ribosome entry sites) that allows the ribosome's 40S subunit to bind directly. Originally identified as a translation mechanism of viral genes, it is now identified as playing an important role during the death cell's process, mitosis, and stress conditions, where cap-dependent protein synthesis is reduced (Stoneley & Willis, 2004 ).
As the 30S complex slides across the mRNA into its preinitiation position, many noncovalent bonds are created and broken. The stability of the preinitiation complex and the translation initiation rate is determined by the energetics of these bonds. The complex's assembly rate is decreased by the unfolding of mRNA structures that sequester the 16S rRNA binding site, spacer region, start codon, or ribosome footprint region ( Studer and Joseph, 2006 ). These mRNA structures are composed of intramolecular nucleotide base pairings (hydrogen bonds) that form helices, knots, loops, and bulges. The absence of these mRNA structures will increase the translation initiation rate. Importantly, both the RBS and protein CDSs can participate in these mRNA structures.
eIF-2 binds GTP, then eIF-2/GTP binds Met-tRNA, then eIF-1 and eIF-2 bind small ribosomal subunit, then eIF-4 guides the the complexes together to the 5' end of mRNA, then the large ribosomal subunit binds
a lipid of the ER membrane that acts as a carrier for the universal sugar as it is being built
stop codon enters A-site, then eRF bound to GTP binds with codon, then the protein chain is released , then GTP is hydrolyzed , then the transcription complex disassociates
adds a phosphate to a hydroxyl group to activate or inactivate the protein
aminoacyl tRNA binds at A-site, then peptidyltransferase of the large ribosomal subunit forms a peptide bond between the adjacent amino acids, then the tRNA translocates to the P-site, then to the E-site
a leader sequence on the N-terminal is synthesized as the protein is being synthesized, then guided into the mitochondria via the TOM/TIM complex, then the leader sequence is cleaved off
postranslational modification of mannose-6-phosphate targets the enzyme to the lysosome