Cloning and characterization of the Neurospora crassa cyt-5 gene. A nuclear-coded mitochondrial RNA polymerase with a polyglutamine repeat.

The Neurospora crassa mutants, cyt-5-1 and cyt-5-4 have a cytochrome b- and aa3-deficient phenotype, suggesting that they result from a deficiency in a nuclear-coded component of the mitochondrial gene expression apparatus (Bertrand, H., Nargang, F. E., Colllins, R. A., and Zagozeski, C. A. (1977) Mol. Gen. Genet. 153,247-257). The complementing wild-type gene has been cloned and and shown to encode a protein with significant sequence similarity to Saccharomyces cerevisiae mitochondrial RNA polymerase and bacteriophage RNA polymerases. There are remarkable differences between the N. crassa protein and its yeast homologue, including a region of very little homology near the N termini of the two gene products. The cyt-5 gene encodes a stretch of polyglutamine in this region of uniquesequence. In addition, an acidic insertion (86 amino acids, of which 24 are Asp or Glu and 10 are Arg or Lys) is present near the C terminus of the cyt-5 gene product. Transcript levels of the cytochrome b and cytochrome oxidase subunit III genes are severely reduced in cyt-5 mutants, suggesting a likely mechanism for the cytochrome-deficient phenotype. In contrast, mitochondrial rRNAs accumulate to nearly normal levels in cyt-5 mutants. However, mitochondrial rRNA levels are not indicative of the rate of transcription of the corresponding genes, since crude lysates of mitochondria from cyt-5 mutants exhibit greatly reduced transcriptional activity with a 19 S rRNA promoter. The cyt-5 gene is flanked by at least one gene whose product also may be involved in mitochondrial function.

Synechocystis sp. PCC6803 fusB gene, located outside of the str operon, encodes a polypeptide related to protein synthesis factor EF-G.

Synechocystis sp. PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon. The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results). There is no evidence for the presence of a fusB homologue in other bacteria. The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes.

Purification and N-terminal sequence analysis of pea chloroplast protein synthesis factor EF-G.

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea (Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

Cloning and sequencing of a soybean nuclear gene coding for a chloroplast translation elongation factor EF-G.

A plant nuclear gene coding for a chloroplast specific translation elongation factor EF-G (cEF-G) was cloned and sequenced for the first time. We screened two partial soybean genomic libraries with a short PCR amplified pea DNA probe constructed according to the N-terminal peptide sequence of pea chloroplast EF-G. The gene is three times split, codes for a chloroplast type transit peptide and a protein very similar to bacterial translation elongation factor EF-G. The gene is expressed as evidenced by Northern hybridisations.

Light regulation of protein synthesis factor EF-G in pea chloroplasts.

The activity of pea chloroplast elongation factor G (EF-G), a nuclear-coded protein required for the elongation cycle of chloroplast protein synthesis, is regulated in response to light. In pea seedlings germinated and grown under continuous white or red light, EF-G specific activity reaches a maximum between days 10 to 15, and then decreases. EF-G activity is almost undetectable in extracts from dark-grown seedlings. When 13-day dark-grown pea seedlings are transferred to light, EF-G specific activity reaches a higher value after 2 to 3 days than observed in seedlings grown under continuous light. The small and large subunits of ribulose bisphosphate carboxylase continue to accumulate after EF-G specific activity has reached maximum levels. Cytoplasmically synthesized components of the chloroplast protein synthetic apparatus, such as EF-G, may help coordinate cytoplasmic and nuclear events with chloroplast gene expression during light-induced chloroplast differentiation.

The E35 stopper mutant of Neurospora crassa: precise localization of deletion endpoints in mitochondrial DNA and evidence that the deleted DNA codes for a subunit of NADH dehydrogenase.

Two types of defective mitochondrial DNA molecules with large deletions (5 kbp and 40 kbp) have previously been identified in the stopper mutant, E35, of Neurospora crassa. The junction fragments spanning the deletion endpoints have now been cloned and sequenced, and their sequences compared with those of the corresponding wild-type fragments. We show that both types of defective mitochondrial DNAs result from deletions of sequences flanked by short direct repeats, which are themselves parts of larger inverted repeat sequences. In every case, the short direct repeat sequences consist of a run of pyrimidines in one strand and purines in the other. We also report the sequence of a 2151-bp HindIII fragment, which is deleted in both of the defective mitochondrial DNAs. Besides the previously identified gene for a methionine tRNA, the 2151-bp DNA sequence contains an open reading frame with the potential to code for a hydrophobic protein 583 amino acids long. This hydrophobic protein has three blocks of significant homology with proteins coded by URF2 found in other mitochondrial genomes. Since the mammalian mitochondrial URF2 has recently been shown to code for a subunit of NADH dehydrogenase, part of the DNA sequence missing in the E35 stopper mutant of N. crassa may also code for a subunit of NADH dehydrogenase.

RNA processing in Neurospora crassa mitochondria: use of transfer RNA sequences as signals.

We have used RNA gel transfer hybridization, S1 nuclease mapping and primer extension to analyze transcripts derived from several genes in Neurospora crassa mitochondria. The transcripts studied include those for cytochrome oxidase subunit III, 17S rRNA and an unidentified open reading frame. In all three cases, initial transcripts are long, include tRNA sequences, and are subsequently processed to generate the mature RNAs. We find that endpoints of the most abundant transcripts generally coincide with those of tRNA sequences. We therefore conclude that tRNA sequences in long transcripts act as primary signals for RNA processing in N. crassa mitochondria. The situation is somewhat analogous to that observed in mammalian mitochondrial systems. The difference, however, is that in mammalian mitochondria, noncoding spacers between tRNA, rRNA and protein genes are very short and in many cases non-existent, allowing no room for intergenic RNA processing signals whereas, in N. crassa mtDNA, intergenic non-coding sequences are usually several hundred nucleotides long and contain highly conserved GC-rich palindromic sequences. Since these GC-rich palindromic sequences are retained in the processed mature RNAs, we conclude that they do not serve as signals for RNA processing.

Purification of Euglena gracilis chloroplast elongation factor G and comparison with other prokaryotic and eukaryotic translocases.

Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.