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.     

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.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP

The crystal structure of bovine mitochondrial elongation factor Tu (EF-Tu) in complex with GDP has been determined at a resolution of 1. 94 A. The structure is similar to that of EF-Tu:GDP from Escherichia coli and Thermus aquaticus, but the orientation of the GDP-binding domain 1 is changed relative to domains 2 and 3. Sixteen conserved water molecules common to EF-Tu and other G-proteins in the GDP-binding site are described. These water molecules create a network linking separated parts of the binding pocket. Mitochondrial EF-Tu binds nucleotides less tightly than prokaryotic EF-Tu possibly due to an increased mobility in regions close to the GDP-binding site. The C-terminal extension of mitochondrial EF-Tu has structural similarities with DNA recognising zinc fingers suggesting that the extension may be involved in recognition of RNA.     

Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed.     

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).     

Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E. coli EF-Tu

The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E. coli have been investigated. The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E. coli EF-Ts. The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E. coli EF-Ts. The E. coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E. coli EF-Tu:E. coli EF-Ts complex.     

Splicing error in E1alpha pyruvate dehydrogenase mRNA caused by novel intronic mutation responsible for lactic acidosis and mental retardation

An intronic point mutation was identified in the E1alpha PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the alpha and beta subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1alpha PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5'-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5'-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Nova regulates GABA(A) receptor gamma2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer

Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABA(A) receptor gamma2 (GABA(A)Rgamma2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA(A)Rgamma2 alternative splicing, we systematically screened minigenes derived from the GABA(A)Rgamma2 and human beta-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA(A)Rgamma2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABA(A)Rgamma2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.     

Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.