D-E-A-D protein family of putative RNA helicases

RNA metabolism plays a central role in cell growth. It is essential to regulate RNA synthesis, processing, stability and degradation. Conformational changes in RNA are key elements in regulating cellular processes. Recently, an increasing number of putative RNA helicases from different organisms ranging from Escherichia coli to humans and viruses have been identified. They are involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division. Based on sequence homologies these proteins were grouped in a family, the D-E-A-D box protein family (D-E-A-D = Asp-Glu-Ala-Asp). Some of the better characterized members have been shown to possess ATP-binding and hydrolysing activities as well as ATP-dependent RNA helicase activities. Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review. From sequence data, three of the members form a subfamily, the D-E-A-H subfamily.     

deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.     

Dbp73D, a Drosophila gene expressed in ovary, encodes a novel D-E-A-D box protein.

Proteins of the D-E-A-D family of putative ATP-dependent RNA helicases have been implicated in translation initiation and RNA splicing in a variety of organisms from E. coli to man. The Drosophila vasa protein, a member of this family, is required in the female germ line for fertility and for specification of germ line and posterior positional information in progeny embryos. We report the isolation of another D-E-A-D gene from Drosophila, which, like vasa, is expressed in germ line tissue. The predicted amino acid sequence of this new gene, Dbp73D, contains all of the highly conserved helicase motifs, but is otherwise the farthest-diverged member of the family so far identified.     

Metagenomic DNA fragments that affect Escherichia coli mutational pathways

A multicopy cloning approach was used to search for metagenomic DNA fragments that affect Escherichia coli mutational pathways. Soil metagenomic expression libraries were constructed with DNA samples prepared directly from soil samples collected from the UCLA Botanical Garden. Using frameshift mutator screening, we obtained a total of 26 unique metagenomic fragments that stimulate frameshift rates in an E. coli wild-type host. Mutational enhancer strains such as an ndk-deficient strain and a temperature sensitive mutS strain (mutS60) were used to further verify the mutator phenotype. We found that the presence of multiple copies of certain types of metagenomic DNA sequence repeats cause general genome instability in the wild-type E. coli host and the effect can be suppressed by overproducing a DNA mismatch component MutL. In addition, we identified nine metagenomic mutator genes (designated as smu genes) that encode proteins that have not been linked to mutator phenotypes prior to this study including a putative RNA methyltransferase Smu10A. The strain overproducing Smu10A displays one prominent base substitution hotspot in the rpoB gene, which coincides with the base substitution hotspot we have observed in cells that are partially deficient in the proofreading function carried out by the DNA polymerase III epsilon subunit. Based on the structural conservation of DNA replication/recombination/repair machineries among microorganisms, this approach would allow us to both identify new mutational pathways in E. coli and to find genes involved in DNA replication, recombination or DNA repair from vast unculturable microbes.     

Identification of a putative RNA helicase in E.coli.

The human p68 protein, an SV40 large T related antigen, is an RNA dependent ATPase and RNA helicase. It belongs to a new large and highly conserved gene family, the DEAD box proteins, whose members are involved in a variety of processes requiring manipulation of RNA secondary structure such as translation and splicing. Multiple DEAD box genes are present in S.cerevisiae, but only one has previously been described in E.coli. Low stringency screening of an E.coli genomic library with a p68 cDNA probe led to the identification of dbpA, a new E.coli DEAD box gene located at 29.6 minutes on the W3110 chromosome. We report here the nucleotide and deduced amino acid sequences of the gene. We have overexpressed dbpA from its own promoter on a high copy number plasmid and identified the gene product as a approximately 50 kD protein by immunoblotting with an anti-DEAD antibody.     

The DEAD box proteins DDX5 (p68) and DDX17 (p72): Multi-tasking transcriptional regulators

Members of the DEAD box family of RNA helicases, which are characterised by the presence of twelve conserved motifs (including the signature D-E-A-D motif) within a structurally conserved ‘helicase’ core, are involved in all aspects of RNA metabolism. Apart from unwinding RNA duplexes, which established these proteins as RNA helicases, DEAD box proteins have been shown to also catalyse RNA annealing and to displace proteins from RNA. DEAD box proteins generally act as components of large multi-protein complexes and it is thought that interactions, via their divergent N- and C-terminal extensions, with other factors in the complexes may be responsible for the many different functions attributed to these proteins.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.     

微生物产生的冷休克蛋白研究进展

冷休克蛋白(cold shock protein,Csp)首先在大肠杆菌中发现,它与微生物对冷环境的适应及多种细胞功能有关。冷休克蛋白基因是一段编码70个左右氨基酸的DNA序列,在这段序列中有5′非翻译区(5′UTR)、冷盒及下游盒等特征。冷休克蛋白作为DNA或RNA结合蛋白在基因表达调控过程中起重要作用。冷休克蛋白在转录、mRNA稳定性及翻译等几个水平上被严格调控。     

M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies.     

The priB and priC replication proteins of Escherichia coli. Genes, DNA sequence, overexpression, and purification.

The Escherichia coli DNA replication proteins n and n" function in vitro in the assembly of the primosome, a mobile multiprotein replication priming complex thought to operate on the lagging-strand template at the E. coli DNA replication fork. Both proteins have been purified from E. coli HMS83 cells based on their requirement for the reconstitution of bacteriophage phi X174 complementary strand DNA synthesis in vitro with purified proteins. As a step toward understanding the role of these proteins in vivo, the genes for primosomal proteins n and n", designated priB and priC, respectively, have been cloned molecularly. priB encodes a 104-amino acid 11.4-kDa polypeptide and corresponds to an previously identified open reading frame between rpsF and rps R within a ribosomal protein operon at 95.5 min on the E. coli chromosome. priC encodes a 175-amino acid 20.3-kDa polypeptide. These two gene products were overexpressed at least 1000-fold in E. coli using a bacteriophage T7 transient expression system. Both proteins have been purified to apparent homogeneity from extracts prepared from these overproducing strains.     

Sequence of a putative promoter region for the rRNA genes of tobacco chloroplast DNA.

The nucleotide sequence of the segment of tobacco chloroplast DNA adjacent to and including the start of the 16S rRNA gene has been determined. The region just preceding this gene was found to contain a tRNAVal gene and promoter-type sequences similar to those which occur in E. coli were found before this tRNA gene. E. coli RNA polymerase can recognize these sequences and in vitro co-transcribes the tRNA and rRNA genes.     

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis

The 3.6 kb HindIII DNA fragment of B. sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51"). DNA fragments carrying only one of these two genes when expressed in E. coli yield products that are inactive towards Culex larvae. The larvicidal activity is recovered when Triton X-100 treated E. coli cells containing each one of the two genes are incubated together. In E. coli these two polypeptides are acting synergistically. The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity. In B. subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42". There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes.     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. I. Structural and functional analysis

Three recombinant plasmids pPBT9, pPBT10 and pPBT74 carrying promoter-containing regions of DNA of Bacillus thuringiensis which are responsible for the expression of the promoterless tet gene, were studied. In the in vitro experiments, it had been shown that these promoter-active HindIII fragments of bacillar DNA contained RNA polymerase binding sites. The AluI subfragments that specifically bind to Escherichia coli RNA polymerase promote the tet gene expression, similar to the whole HindIII fragments. Sequence analysis revealed that the approximately 220 base pair AluI subfragment of the bacillar insertion of the pPBT10 plasmid contained sites typical for "-10" and "-35" homology regions of promoters specific for sigma 55-RNA polymerase from Bac. subtilis. The 1.45 kb HindII bacillar fragment of the plasmid pPBT9 had three AluI subfragments that bind to E. coli RNA polymerase. Only approximately 400 base pair AluI subfragment among these restored the tet gene expression in vivo. Bireplicon pBP plasmids were constructed that promoted the expression of the enterobacterial antibiotic resistance gene under the control of Bac. thuringiensis promoters in Bac. subtilis cells.