Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation factor 3

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) promotes initiation complex formation on mitochondrial 55S ribosomes in the presence of IF2(mt), fMet-tRNA and poly(A,U,G). The mature form of IF3(mt) is predicted to be 247 residues. Alignment of IF3(mt) with bacterial IF3 indicates that it has a central region with 20–30% identity to the bacterial factors. Both the N- and C-termini of IF3(mt) have extensions of ∼30 residues compared with bacterial IF3. To examine the role of the extensions on IF3(mt), deletion constructs were prepared in which the N-terminal extension, the C-terminal extension or both extensions were deleted. These truncated derivatives were slightly more active in promoting initiation complex formation than the mature form of IF3(mt). Mitochondrial 28S subunits have the ability to bind fMet-tRNA in the absence of mRNA. IF3(mt) promotes the dissociation of the fMet-tRNA bound in the absence of mRNA. This activity of IF3(mt) requires the C-terminal extension of this factor. Mitochondrial 28S subunits also bind mRNA independently of fMet-tRNA or added initiation factors. IF3(mt) has no effect on the formation of these complexes and cannot dissociate them once formed. These observations have lead to a new model for the function of IF3(mt) in mitochondrial translational initiation

Role of the conserved aspartate and phenylalanine residues in prokaryotic and mitochondrial elongation factor Ts in guanine nucleotide exchange

AbstractThe guanine nucleotide exchange reaction catalyzed by elongation factor Ts is proposed to arise from the intrusion of the side chains of D80 and F81 near the Mg2+ binding site in EF-Tu. D80A and F81A mutants of E. coli EF-Ts were 2–3-fold less active in promoting GDP exchange with E. coli EF-Tu while the D80AF81A mutant was nearly 10-fold less active. The D84 and F85 mutants of EF-Tsmt were 5–10-fold less active in stimulating the activity of EF-Tumt. The double mutation completely abolished the activity of EF-Tsmt

Roles of Residues in Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Mitochondrial and Bacterial Elongation Factor Tu

The crystal structure of the complex between Escherichia coli elongation factors Tu and Ts (EF-Tu.Ts) and subsequent mutagenesis work have provided insights into the roles of a number of residues in E. coli EF-Ts in its interaction with EF-Tu. The corresponding residues in bovine mitochondrial EF-Ts (EF-Tsmt) have been mutated. The abilities of the resulting EF-Tsmt derivatives to stimulate the activities of both E. coli and mitochondrial EF-Tu have been tested. Mutation of several residues in EF-Tsmt corresponding to amino acids important for the activity of E. coli EF-Ts has little or no effect on the activity of the mitochondrial factor, suggesting that these factors may use somewhat different mechanisms to promote guanine nucleotide exchange. In general, mutations that reduce the strength of the interaction between EF-Tsmt and E. coli EF-Tu increase the ability of EF-Tsmt to stimulate the activity of the bacterial factor. In contrast, these mutations tend to reduce the ability of EF-Tsmt to stimulate the activity of EF-Tumt. For example, F19A/I20A and H176A derivatives of EF-Tsmt are as active as E. coli EF-Ts in simulating E. coli EF-Tu. However, these mutations significantly decrease the ability of EF-Tsmt to stimulate EF-Tumt

Expression, Purification, and Mechanistic Studies of Bovine Mitochondrial Translational Initiation Factor 2

A complete cDNA clone encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained. The regions of the cDNA corresponding to mature IF-2mt and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor (approximately 90 kDa) and mature (approximately 85 kDa) forms of IF-2mt are toxic to E. coli and can only be expressed at low levels. Shorter forms of this factor (approximately 80 and approximately 72 kDa) are also found during the expression of mature IF-2mt. The various forms of IF-2mt can be separated by high performance liquid chromatography. All of these forms are active in promoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovine mitochondrial ribosomes. IF-2mt can bind to mitochondrial ribosomes in the absence of GTP, initiator tRNA, or messenger RNA. The presence of GTP stimulates IF-2mt binding to ribosomes about 3-fold. IF-2mt interacts only weakly with GTP or with the initiator tRNA in the absence of ribosomes. Molecular dissection of IF-2mt shows that a long deletion (approximately 150 amino acid residues) from the NH2-terminal region does not affect its activity in vitro. The COOH domain of IF-2mt (amino acid residues 332-727) can bind to ribosomes even though it does not promote initiator-tRNA binding

Analysis of the translational initiation region on the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase (rbcL) messenger RNA

The chloroplast mRNAs from Euglena gracilis fall into two classes. One group of mRNAs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region. To investigate the start signals for E. gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 90 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/ oxygenase (rbcL) or mutated derivatives of this site

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

Human mitochondrial translational initiation factor 3 (IF3(mt)) has been identified from the human expressed sequence tag data base. Using consensus sequences derived from conserved regions of the bacterial IF3, several partially sequenced cDNA clones were identified, and the complete sequence was assembled in silico from overlapping clones. IF3(mt) is 278 amino acid residues in length. MitoProt II predicts a 97% probability that this protein will be localized in mitochondria and further predicts that the mature protein will be 247 residues in length. The cDNA for the predicted mature form of IF3(mt) was cloned, and the protein was expressed in Escherichia coli in a His-tagged form. The mature form of IF3(mt) has short extensions on the N and C termini surrounding a region homologous to bacterial IF3. The region of IF3(mt) homologous to prokaryotic factors ranges between 21-26% identical to the bacterial proteins. Purified IF3(mt) promotes initiation complex formation on mitochondrial 55 S ribosomes in the presence of mitochondrial initiation factor 2 (IF2(mt)), [(35)S]fMet-tRNA, and either poly(A,U,G) or an in vitro transcript of the cytochrome oxidase subunit II gene as mRNA. IF3(mt) shifts the equilibrium between the 55 S mitochondrial ribosome and its subunits toward subunit dissociation. In addition, the ability of E. coli initiation factor 1 to stimulate initiation complex formation on E. coli 70 S and mitochondrial 55 S ribosomes was investigated in the presence of IF2(mt) and IF3(mt)

Mechanism of protein biosynthesis in mammalian mitochondria

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided

Quantitation of cation binding to wheat germ ribosomes: Influences on submit association equilibria and ribosome activity

The binding of M

Effect of the secondary structure in the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase messenger RNA on translational initiation.

The results reported in the previous paper indicate that the translational start site of the Euglena gracilis chloroplast mRNA for the large subunit of ribulose-bisphosphate carboxylase/oxygenase (rbcL) is not defined by primary sequence elements (Koo, J.S., and Spremulli, L.L. (1994) J. Biol. Chem. 269, 7494-7500). In the work presented here, the effects of secondary structure in the 5'-untranslated leader of the rbcL mRNA have been examined. Only weak secondary structure can be detected in the 5'-untranslated leader of the rbcL message by enzymatic and computer analysis. Further reduction of the weak secondary structure of this message by site-directed mutagenesis does not significantly affect the ability of this message to participate in initiation complex formation. The secondary structure near the translational start site was increased by the introduction of an inverted repeat sequence and by site-directed mutagenesis. Messages with increased secondary structure are much less active in initiation complex formation if the structural element introduced is within approximately 10 nucleotides of the start codon. These results suggest that the translational start site in this chloroplast mRNA is specified by the presence of an AUG codon in an unstructured or weakly structured region of the mRNA. No specific sequences around the start codon, either upstream or immediately downstream, were found to have important information directing the chloroplast ribosome to the start site of this mRNA

Interaction of Mitochondrial Elongation Factor Tu With Aminoacyl-tRNA and Elongation Factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu·GTP·aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu·GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu·Ts). Mammalian mitochondrial EF-Tu (EF-Tumt) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tumt subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K tRNA) and for the dissociation of the EF-Tu·Tsmt complex (K Ts) were investigated. The equilibrium dissociation constant for the ternary complex was 18 ± 4 nM, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 × 10− 4 s− 1, which is essentially equivalent to that observed for the ternary complex inEscherichia coli. The binding of EF-Tumt to EF-Tsmt is mutually exclusive with the formation of the ternary complex. K Ts was determined by quantifying the effects of increasing concentrations of EF-Tsmt on the amount of ternary complex formed with EF-Tumt. The value obtained for K Ts(5.5 ± 1.3 nM) is comparable to the value ofK tRNA