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L L Spremulli

Publications and source records attributed to L L Spremulli.

At least 55 records · Page 3Linked to original sources
PubMed1994

Analysis of the role of the Shine-Dalgarno sequence and mRNA secondary structure on the efficiency of translational initiation in the Euglena gracilis chloroplast atpH mRNA.

Chloroplast mRNAs in Euglena gracilis fall into two classes. One class has a Shine-Dalgarno sequence 5' to the AUG start codon while the other group of mRNAs does not have any conserved sequence elements near the start codon. The chloroplast mRNA encoding the atpH gene has been selected as an example of a message which has a Shine-Dalgarno sequence (GGAGUU) located in the initiation region. Mutations in the Shine-Dalgarno sequence result in 2-5-fold reductions in the efficiency of the message in initiation complex formation depending on the precise mutation. Secondary structure mapping of the initiation region of the atpH mRNA suggests that a number of mRNA conformations are present in an equilibrium mixture. Mutations in the Shine-Dalgarno sequence had little effect on the overall structure of the mRNA. Directed mutations which place the Shine-Dalgarno sequence into a region of higher secondary structure drastically reduce the activity of the message. The monocistronic form of the atpH mRNA has a 5'-untranslated leader region slightly over 70 nucleotides in length. Deletions in the leader suggest that a minimum length of 10 bases 5' to the start codon is important for activity in initiation. The presence of the full leader increased efficiency in initiation about 2-fold compared to messages with leaders 25-40 residues in length.

Animals↗
PubMed1994

Isolation and characterization of cDNA clones for chloroplast translational initiation factor-3 from Euglena gracilis.

A complete cDNA clone encoding Euglena gracilis chloroplast translational initiation factor 3 (IF-3chl) has been obtained. Analysis of the sequence indicates that the IF-3chl mRNA contains the spliced leader found at the 5' end of nuclear encoded mRNAs in E. gracilis. The open reading frame for IF-3chl encodes a 537-amino acid protein. IF-3chl appears to be divided into four domains. The first 140 amino acids correspond to a transit peptide required for the import of IF-3chl into the chloroplast. The mature form of IF-3chl encompasses domains 2-4 and is about twice the size of Escherichia coli IF-3. The second domain has no homology to other known proteins. It begins with a stretch of 35 residues, of which about 30% are proline. Downstream from this region is a stretch of about 25 amino acids with a repeating (GX)n motif followed by a very acidic region. The third domain comprises a region of about 175 residues and has between 31 and 37% homology to the IF-3s found in other organisms. The IF-3 homology domain is followed by an acidic region which has no detectable homology to other sequences. Analysis of E. gracilis genomic DNA suggests that there are about four copies of the IF-3chl gene, one of which is probably a pseudogene. The activity of IF-3chl is inducible by light. However, the IF-3chl mRNA is present in approximately equal amounts in both dark- and light-grown cells, suggesting that the light-dependent induction of IF-3chl activity is post-transcriptional.

Amino Acid Sequence↗
PubMed1994

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 mRNs 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, 30 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. Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon. The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity. Maximal activity requires the full 55-base leader sequence. Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation. Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold. The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site. Similar results are obtained when initiation complexes are formed with initiation factors from either E. gracilis chloroplasts or Escherichia coli.

Animals↗
PubMed1994

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.

Animals↗
PubMed1992

Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa. Subunits of 97 to greater than 200 kDa have been observed in these preparations. Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl. Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related. Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl. Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein. Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis. The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors. Neither polyclonal nor monoclonal antibodies against E. gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.

Animals↗
PubMed1991

Initiation of protein synthesis in animal mitochondria. Purification and characterization of translational initiation factor 2.

Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity. The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity. SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa. IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative. IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes. IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide. It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly. The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP. DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP. No activity is observed with ATP, CTP, or UTP. Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.

Animals↗
PubMed1991

Interactions of bovine mitochondrial phenylalanyl-tRNA with ribosomes and elongation factors from mitochondria and bacteria.

A homologous in vitro poly(U)-directed translation system has been established using animal mitochondrial ribosomes, elongation factors (EF) and phenylalanyl-tRNA(Phe). The rate of incorporation of phenylalanine into polyphenylalanine in the mitochondrial system is slower than that observed for the homologous Escherichia coli system. E. coli ribosomes can be used in place of mitochondrial ribosomes in this system with only a slight decrease in the efficiency of phenylalanine incorporation from mitochondrial Phe-tRNA. However, E. coli elongation factor Tu (EF-Tu) cannot replace the mitochondrial EF-Tu in promoting the use of mitochondrial Phe-tRNA. The interaction between EF-Tu and mitochondrial Phe-tRNA was investigated by using the ability of EF-Tu to protect the aminoacyl-tRNA bond from hydrolysis. These results showed that both mitochondrial and E. coli EF-Tus are capable of interacting with mitochondrial Phe-tRNA. However, ribosomal A-site binding assays demonstrated that efficient binding of the mitochondrial Phe-tRNA to the ribosomal A-site was only obtained with the homologous mitochondrial EF-Tu.

Animals↗
PubMed1991

Chloroplast translational initiation factor 3. Purification and characterization of multiple forms from Euglena gracilis.

The chloroplast translational initiation factor 3 (IF-3chl) has been purified by a combination of gravity and high pressure liquid chromatographic steps. IF-3chl activity has been resolved into three forms designated alpha, beta, and gamma. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the alpha form corresponds to a single polypeptide with a molecular mass of approximately 34 kDa. The beta and gamma forms have been purified to near homogeneity, and both forms appear to function as monomers with molecular masses of about 39-42 kDa. All three forms are heat stable. All the forms of IF-3chl detected enhance the poly (A,U,G)-dependent binding of the initiator tRNA to chloroplast 30 S ribosomal subunits in the presence of Escherichia coli IF-1 and IF-2. The chloroplast factor, unlike the corresponding bacterial factor, does not have a strong RNA binding activity.

Animals↗
PubMed1991

Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes.

The mammalian mitochondrial complex consisting of elongation factors EF-Tu and EF-Ts (EF-Tu.Tsmt) is capable of efficiently binding aminoacyl-tRNA to the ribosome in the presence and absence of guanine nucleotides. In the presence of GTP the binding reaction is catalytic. In the absence of guanine nucleotides, or in the presence of a non-hydrolyzable GTP analog, only one round of ribosome binding occurs. EF-Tu.Tsmt is capable of forming a ternary complex with GTP and Escherichia coli Phe-tRNA as demonstrated by gel filtration chromatography, nitrocellulose filter binding, and by protection of the aminoacyl-tRNA bond from hydrolysis. GDP and the non-hydrolyzable GTP analog guanyl-5'-yl imidodiphosphate are also capable of facilitating ternary complex formation with EF-Tu.Tsmt, but are less effective. No kinetic advantage results from the formation of this ternary complex prior to ribosome binding, and EF-Tu.Tsmt may actually bind aminoacyl-tRNA directly to the ribosome prior to binding GTP. These results suggest that a variation of the prokaryotic elongation cycle is occurring in animal mitochondria. N-Ethylmaleimide inhibits the activity of EF-Tu.Tsmt in polymerization and in ribosome binding. However, the activity of the EF-Tsmt which can be measured independently, is not altered.

Animals↗
PubMed1990

Purification and characterization of elongation factor G from bovine liver mitochondria.

The mitochondrial protein synthesis translocase elongation factor Gmt (EF-Gmt) from bovine liver has been purified to greater than 90% homogeneity by a combination of conventional gravity and high performance liquid chromatography. The purification scheme results in an approximate overall 14,000-fold purification with 2% total recovery of EF-Gmt activity. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the mitochondrial factor is a single polypeptide with a molecular weight of 80,000. EF-Gmt displays similar levels of activity on its homologous mitochondrial ribosomes and on Escherichia coli ribosomes. The mitochondrial translocase is sensitive to temperatures above 37 degrees C, but the factor is partially protected from heat inactivation in the presence of GTP or GDP. The activity of EF-Gmt is inhibited by treatment of the factor with N-ethylmaleimide. In contrast to all other translocases tested to date, EF-Gmt is completely resistant to the inhibiting effect of fusidic acid when tested on its homologous ribosomes. It displays weak sensitivity to this antibiotic when assayed in the presence of heterologous E. coli ribosomes.

Animals↗
PubMed1990

Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa. The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose. Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight. In addition, one form consisting of subunits of about 200,000 Mr has been detected. All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction. Initiation complex formation promoted by IF-2chl requires the presence of GTP. Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used. The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3. None of the forms of IF-2chl detected is active on E. coli ribosomes.

Animals↗
PubMed1990

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria.

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.

Animals↗
PubMed1990

Effects of length and mRNA secondary structure on the interaction of bovine mitochondrial ribosomes with messenger RNA.

The mRNA for cytochrome oxidase subunit II (CoII) from bovine mitochondria binds to the small subunit of the mitochondrial ribosome in the absence of auxiliary factors. The synthetic polymer poly(U) is effective in competing with CoII mRNA for binding, although the polymer poly(A,U,G) competes very weakly. The effects of mRNA length on the interaction between the 28 S ribosomal subunit and mRNA have been examined using truncated derivatives of CoII mRNA. These results indicate that there is a minimum length of approximately 400 nucleotides required for the efficient binding of the mRNA to the small subunit. Shorter mRNAs will bind, but do so with much lower association constants. mRNAs of various lengths but with reduced secondary structure were prepared by substituting ITP for GTP during in vitro transcription reactions. These derivatives show the same effects of length as do the normal mRNA, indicating that RNA secondary structure is not a critical factor in subunit-mRNA interaction. The binding of the mRNA to the 28 S subunit is not influenced by the presence of guanine nucleotides or by the presence of a triphosphate at the 5' end of the RNA.

Animals↗
PubMed1990

Generation of potential structures for the G-domain of chloroplast EF-Tu using comparative molecular modeling.

Comparative molecular modeling has been used to generate several possible structures for the G-domain of chloroplast elongation factor Tu (EF-Tu(chl)) based on the crystallographic data of the homologous E. coli protein. EF-Tu(chl) contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF-Tu(chl) have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation state of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF-Tu(chl) have been obtained. The interactions between EF-Tu(chl) and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.

Chloroplasts↗
PubMed1989

Initiation complex formation on Euglena chloroplast 30S subunits in the presence of natural mRNAs.

An in vitro system has been developed that allows the formation of translation initiation complexes with Euglena chloroplast 30S ribosomal subunits and natural mRNAs. For these experiments two regions of the Euglena chloroplast genome have been cloned behind the T7 transcriptional promoter and the corresponding RNAs synthesized in vitro. These mRNAs are capable of forming initiation complexes with chloroplast 30S subunits in the presence of fMet-tRNA and E. coli initiation factors. Deletion of the normal translation start site results in a message that is no longer recognized by the chloroplast subunits suggesting that the correct AUG initiation codon on the mRNA is being selected by the small ribosomal subunit. Initiation complex formation with the chloroplast 30S subunits is specific for chloroplast mRNAs and mRNA from the phage MS2 is not active in this system.

Animals↗
PubMed1989

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex.

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.

Animals↗
PubMed1989

Interaction of bovine mitochondrial ribosomes with messenger RNA.

The gene for subunit II of cytochrome oxidase (CoII) from bovine mitochondria has been cloned behind a T7 promoter and the corresponding mRNA synthesized in vitro. The RNA transcribed from this vector has a single nucleotide 5' to the start AUG and, thus, corresponds closely to the native mRNA. It binds to the small 28 S ribosomal subunit of bovine mitochondria but not to the large (39 S) subunit or to 55 S ribosomes. The binding occurs readily in the absence of auxiliary initiation factors or initiator tRNA. The complex formed appears to contain 1 mRNA/28 S subunit. The observed binding is specific for mRNA since neither tRNA nor ribosomal RNA can act as competitive inhibitors. The interaction of the mRNA with the 28 S subunit does not require an AUG codon near the 5' end and constructs containing 5' leaders of more than 100 nucleotides still bind efficiently. About 5% of the bound mRNA is protected from digestion by T1 RNase. The protected fragments do not arise from a specific region of the mRNA since they hybridize to several restriction fragments of the cloned CoII gene.

Animals↗