Biomedical subjects

L L Spremulli

Publications and source records attributed to L L Spremulli.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regulation of the activity of chloroplast translational initiation factor 3 by NH2- and COOH-terminal extensions.

The mature form of the chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions. Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal subunits. A series of deletions of the NH2- and COOH-terminal extensions of IF3chl were constructed and tested for their effects on the activity of the homology domain. About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain. The remaining inhibitory effect is the result of sequences in the COOH-terminal extension. The equilibrium constant governing the binding of the homology domain of IF3chl to 30 S subunits is estimated to be 1.3 x 10(7) M-1. Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold. Sequences in the COOH-terminal extension have a similar negative effect. The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant. The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3chl. The entire COOH-terminal extension reduces the proofreading ability by about half. These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3chl.

Animals↗

PubMed1997

Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis.

Chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis contains a central region (homology domain) that is homologous to prokaryotic IF3. The homology domain is preceded by a long NH2-terminal extension (head), and followed by a 64 amino acid COOH-terminal extension (tail). Sequences in these extensions reduce the activity of the homology domain. To gain insight into these effects, a possible three-dimensional structure for the homology region of IF3chl has been modeled using the X-ray coordinates from the N- and C-domains of Bacillus stearothermophilus IF3. In B. stearothermophilus IF3, these two compact domains are thought to fold independently and are separated by a helical lysine-rich linker. The modeled structure suggests that IF3chl has a similar overall fold although some subtle differences are predicted to occur. Both the head and tail regions of IF3chl are oriented toward the linker region in the homology domain where they may potentially interfere with its function. Circular dichroism spectropolarimetry (CD) indicates that the lysine-rich linker region in IF3chl is not in a helical conformation and is probably a random coil. CD analysis indicates that a portion of the tail region of IF3chl is helical and that the tail has a direct interaction with the linker region in the homology domain. Site-directed mutagenesis of the linker indicates that two conserved lysine residues are important for the function of IF3chl and play a role in the binding of IF3chl to the 30S ribosomal subunit. Mutation of these residues affects the interaction of the homology domain with the tail.

Amino Acid Sequence↗

PubMed1997

Role of domains in Escherichia coli and mammalian mitochondrial elongation factor Ts in the interaction with elongation factor Tu.

Bovine mitochondrial elongation factor Ts (EF-Tsmt) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu). In contrast, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. EF-Tsmt forms a tight complex with E. coli EF-Tu governed by an association constant of 8.6 x 10(10). This value is 100-fold stronger than the binding constant for the formation of the E. coli EF-Tu.Ts complex. To test which domain of EF-Tsmt is important for its strong binding with EF-Tu, chimeras were made between E. coli EF-Ts and EF-Tsmt. Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases its binding to E. coli EF-Tu 2-3-fold. Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E. coli EF-Ts decreases its binding to E. coli EF-Tu approximately 4-5-fold. A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Tsmt binds to E. coli EF-Tu as strongly as EF-Tsmt. A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EF-Tsmt binds E. coli EF-Tu approximately 25-fold more tightly than E. coli EF-Ts. Thus, the higher strength of the interaction between EF-Tsmt and EF-Tu can be localized primarily to a single subdomain.

Animals↗

PubMed1997

Mechanistic studies of the translational elongation cycle in mammalian mitochondria.

Polyclonal antibodies have been prepared against both components of the bovine liver mitochondrial translational elongation factor Tu and Ts complex (EF-Tu x Ts(mt)). The antibodies against EF-Tu(mt) cross-react somewhat with Escherichia coli EF-Tu and wheat germ EF-1alpha. The antibodies against EF-Ts(mt) cross-react little, if at all, with E. coli EF-Ts or with EF-Ts from Euglena gracilis chloroplasts. These polyclonal antibodies have been used to investigate the relative amounts of EF-Tu(mt) and EF-Ts(mt) in bovine liver mitochondria and in cultured cells. The results of this analysis suggest that there is a 1:1 ratio of EF-Tu(mt) to EF-Ts(mt) in mammalian mitochondria. Intermediate complexes formed during the elongation cycle of protein synthesis in bovine liver mitochondria have also been investigated. The EF-Tu x Ts(mt) complex is quite resistant to dissociation by guanine nucleotides. This complex will, however, dissociate in the presence of GTP and Phe-tRNA resulting in the formation of a ternary complex comparable to that observed in prokaryotes. Kinetic data suggest that the use of the ternary complex in chain elongation increases the rate of Phe-tRNA binding to ribosomes, suggesting that it is a true intermediate in the elongation cycle. Sucrose gradient analysis indicates that the binding of EF-Tu(mt) to ribosomes can be detected in the presence of Phe-tRNA and a non-hydrolyzable analog of GTP. These results suggest that, in contrast to previous thinking, the basic features of the elongation cycle in mammalian mitochondria are quite similar to those in prokaryotes.

Animals↗

PubMed1997

Expression of bovine mitochondrial elongation factor Ts in Escherichia coli and characterization of the heterologous complex formed with prokaryotic elongation factor Tu.

When bovine mitochondrial elongation factor Ts (EF-Ts(mt)) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli EF-Tu (EF-Tu(Eco) x Ts(mt)). This complex is active in poly(U)-directed polymerization and this activity is inhibited by kirromycin. The EF-Tu(Eco) x Ts(mt) complex does not bind guanine nucleotides detectably and is not dissociated to a significant extent by either GDP or GTP. A portion of the EF-Tu(Eco) x Ts(mt) complex can be dissociated by aa-tRNA in the presence of GTP. The heterologous complex cannot be dissociated completely in the presence of either the 8 M urea or 8 M guanidine hydrochloride, suggesting that EF-Ts(mt) has an unusually tight interaction with E. coli EF-Tu. The EF-Tu(Eco) x Ts(mt) complex can be dissociated by denaturation using 2 M guanidine thiocyanate. Free EF-Ts(mt) can then be purified and renatured. The refolded EF-Ts(mt) is active in stimulating the activity of expressed mitochondrial EF-Tu (EF-Tu(mt)) in poly(U)-directed polymerization. Almost all the EF-Ts(mt) molecules appear to refold into a conformation which can interact with EF-Tu(mt). Protease mapping of EF-Ts(mt) indicates that the first 54 residues fold into an independent domain. Analysis of deletion derivatives of EF-Ts(mt) indicates that extensive regions of this factor are required for its tight interaction with EF-Tu.

Animals↗

PubMed1997

Mitochondrial methionyl-tRNA transformylase from bovine liver.

Substrate specificities of mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) toward tRNA substrates were characterized in vitro. The MTFmt is able to formylate E. coli initiator methionyl-tRNA (Met-tRNA(fMet)) as efficiently as mammalian mitochondrial methionyl-tRNA. Furthermore, E. coli elongator methionyl-tRNA (Met-tRNA(mMet)) also serves as a substrate for mt MTF, whereas E. coli MTF rigorously excludes E. coli Met-tRNA(mMet) from formylation reaction. Thus, mammal mt MTF is suggested to have recognition mechanism different from E. coli MTF. To pursue the relationship between protein structure and unexpected substrate specificity of mammalian MTFmt, the nucleotide sequence of MTFmt gene was determined and its amino acids sequence was compared to other MTFs of prokaryotic origin.

Animals↗

PubMed1996

Analysis of the interaction between bovine mitochondrial 28 S ribosomal subunits and mRNA.

The small subunit of the bovine mitochondrial ribosome forms a tight complex with mRNAs. This [28 S:mRNA] complex forms as readily on circular mRNAs as on linear mRNAs indicating that a free 5' end on the mRNA is not required for the interaction observed. The effects of monovalent cations on the equilibrium association constant and on the forward and reverse rate constants governing this interaction have been determined. Monovalent cations have a strong effect on the forward rate constant. Increasing the KCl concentration from 1 mM to 100 mM reduces kon by nearly 100-fold. Monovalent cations have only a small effect on the reverse rate constant, koff'. Analysis of these data indicates that the rate laws governing the formation and dissociation of the [28 S:mRNA] complex cannot be deduced from the chemical equation. This observation suggests that there are "hidden intermediates' in the formation and dissociation of this complex. The implications of these observations are discussed in terms of a model for the interaction between the mitochondrial 28 S subunit and mRNAs.

Animals↗

PubMed1996

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

The 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.

Aspartic Acid↗

PubMed1996

Nucleotide and aminoacyl-tRNA specificity of the mammalian mitochondrial elongation factor EF-Tu.Ts complex.

The bovine mitochondrial elongation factor Tu.Ts complex (EF-Tu.Tsmt) promotes the binding of aminoacyl-tRNA to ribosomes. In the presence of GTP, this complex functions catalytically. Both dGTP and ddGTP can replace GTP although about 4-fold higher concentrations are required. ATP, CTP and UTP are not active. ITP can replace GTP when used at 10- to 20-fold higher concentrations. The catalytic use of EF-Tu.Tsmt is inhibited by GDP but not by GMP. XDP also inhibits although about 20-fold higher concentrations are required. EF-Tu.Tsmt will promote the binding of Phe-tRNA to either Escherichia coli or mitochondrial ribosomes. Unlike E. coli EF-Tu, EF-Tu.Tsmt will promote the binding of AcPhe-tRNA to ribosomes about 25% as efficiently as Phe-tRNA. EF-Tu.Tsmt is active in catalyzing the binding of E. coli Met-tRNAmmet to ribosomes. EF-Tu.Tsmt has about 30% as much activity with E. coli Met-tRNAimet but has essentially no activity with E. coli fMet-tRNAimet. Neither yeast Met-tRNAimet nor fMet-tRNAimet is recognized by bovine EF-Tu.Tsmt.

Acylation↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3.

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3chlM, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3chlH, molecular mass 22 829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3chlM and IF-3chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3chlM and IF-3chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3chlH has at least 5-fold more activity than either native IF-3chl or IF-3chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3chl lying outside of the homology domain may down-regulate the activity of this factor.

Animals↗

PubMed1996

Bovine mitochondrial initiation and elongation factors.

The procedures summarized above provide nearly homogeneous preparations of IF-2mt, EF-Tu. Tsmt, and EF-Gmt. The scheme developed for IF-2mr leads to a 24,000-fold purification of this factor with a 26% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that this factor functions as a monomer with a molecular weight of about 85,000. The scheme developed EF-Tu.Tsmt provides a 10,000-fold purification with an overall yield of about 10%. The EF-Tumt component in this complex has a molecular weight of about 46,000, whereas EF-Tsmt has a molecular weight of about 32,000 on SDS-polyacrylamide gel electrophoresis. The EF-Tu. Tsmt complex is tightly associated and appears to have a native molecular weight of about 70,000. The five-step purification procedure outlined above for EF-Gmt results in a 14,000-fold purification of EF-Gmt with a 2-5% recovery of activity. Analysis by SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicates that EF-Gmt functions as a monomeric protein with an apparent molecular weight of about 80,000.

Animals↗

PubMed1995

Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu.

The bovine liver mitochondrial protein synthesis elongation factor Tu.Ts complex (EF-TU.Tsmt) has been purified and partial peptide sequence information has been obtained for EF-Tumt. A complete cDNA has been obtained encoding bovine EF-Tumt and a nearly complete cDNA has been obtained for human EF-Tumt. The bovine cDNA has a 5' untranslated leader, an open reading frame of 1356 nucleotides and a 3' untranslated region of 189 base pairs. NH2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length. The human and bovine factors are 95% identical. The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences. At least two genes for EF-Tumt are present in the bovine system. Northern analysis indicates that EF-Tumt is synthesized in all tissues but that the level of expression varies over a wide range. EF-TUmt has been expressed in E. coli as a His-tagged protein and purified to near homogeneity. The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu.Tsmt complex.

Amino Acid Sequence↗

PubMed1995

Cloning and expression of mitochondrial translational elongation factor Ts from bovine and human liver.

The sequences of the cDNAs for the mitochondrial translational elongation factor Ts (EF-Tsmt) from bovine and human liver have been obtained. The deduced amino acid sequence of bovine liver EF-Tsmt is 338 residues in length and includes a 55-amino acid signal peptide and a mature protein of 283 residues. The sequence of the mature form of bovine EF-Tsmt is 91% identical to that of human EF-Tsmt and 29% identical to Escherichia coli EF-Ts. Southern analysis indicates that there are two genes for EF-Tsmt in bovine liver chromosomal DNA. A 224-base pair intron is located near the 5'-end of at least one of these genes. Northern analysis using a human multiple tissue blot indicates that EF-Tsmt is expressed in all tissues, with the highest levels of expression in skeletal muscle, liver, and kidney. Both the mature and precursor forms of bovine liver EF-Tsmt have been expressed in E. coli as histidine-tagged proteins. The mature form of EF-Tsmt forms a complex with E. coli elongation factor Tu. This complex is active in poly(U)-directed polymerization of phenylalanine. The precursor form is expressed as a 42-kDa protein, which is rapidly degraded in the cell.

Amino Acid Sequence↗

PubMed1995

Cloning and sequence analysis of the cDNA for bovine mitochondrial translational initiation factor 2.

The complete sequence of the cDNA encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained by library screening followed by 3'-RACE PCR. The open reading frame for bovine IF-2mt encodes a protein of 727 amino acids. The sequence of bovine IF-2mt exhibits 85% identity to human IF-2mt, but only 38% identity to yeast IF-2mt and 39% identity to Escherichia coli IF-2 alpha.

Animals↗

PubMed1995

Cloning and sequence analysis of the human mitochondrial translational initiation factor 2 cDNA.

Complete cDNAs encoding human mitochondrial translational initiation factor 2 (IF-2mt) have been obtained from liver, heart, and fetal brain cDNA libraries. These cDNAs have a long open reading frame 2181 residues in length encoding a protein of 727 amino acids. Overall, human IF-2mt has 30-40% identity to the corresponding prokaryotic factors. Surprisingly, it is no more homologous to yeast IF-2mt than to the IF-2s from bacterial sources. The greatest region of conservation lies in the G-domain of this factor with less conservation in the COOH-terminal half of the protein and very little homology near the amino terminus. The 5'-untranslated leaders of the liver and heart cDNAs contain a number of short open reading frames. These sequences may play a role in the translational activity of the IF-2mt mRNA. Northern analysis indicates that the IF-2mt gene is expressed in all tissues but that the level of expression varies over a wide range.

Amino Acid Sequence↗

PubMed1995

The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons.

The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt. In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.

Animals↗

PubMed1995

Interaction of mitochondrial elongation factors Tu.Ts with aminoacyl-tRNA.

The interaction between the bovine mitochondrial translational elongation factor Tu.Ts complex (EF-Tu.Tsmt) and aminoacyl-tRNA has been investigated using a nuclease protection assay and fluorescence enhancement of [AEDANS-s2C]Tyr-tRNA(Tyr). The equilibrium dissociation constant, Kd, for the EF-Tu.Tsmt:GTP:E. coli Phe-tRNA complex is approximately 50 nM. A similar binding constant (30 nM) is obtained using bovine mitochondrial Phe-tRNA. The equilibrium binding constant for the EF-Tu.Tsmt:GTP:yeast [AEDANS-s2C]Tyr-tRNA(Tyr) complex is approximately 4 nM when determined using the fluorescence enhancement assay.

Animals↗