Low Levels of RNase H Activity in Escherichia coli FB2 rnh Result from a Single-Base Change in the Structural Gene of RNase H

The DNA coding for RNase H from a mutant strain of Escherichia coli (FB2) was cloned into plasmid pBR322. DNA sequence analysis and the exchange of a portion of the mutant and wild-type genes revealed that a single-base alteration (C-->T) in the coding region of the structural gene for RNase H is responsible for the difference in RNase H activity of the wild-type and mutant cells.     

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.     

Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8.

Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.     

Identification of the gene encoding a type 1 RNase H with an N-terminal double-stranded RNA binding domain from a psychrotrophic bacterium

The gene encoding a bacterial type 1 RNase H, termed RBD-RNase HI, was cloned from the psychrotrophic bacterium Shewanella sp. SIB1, overproduced in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RBD-RNase HI consists of 262 amino acid residues and shows amino acid sequence identities of 26% to SIB1 RNase HI, 17% to E. coli RNase HI, and 32% to human RNase H1. SIB1 RBD-RNase HI has a double-stranded RNA binding domain (RBD) at the N-terminus, which is commonly present at the N-termini of eukaryotic type 1 RNases H. Gel mobility shift assay indicated that this domain binds to an RNA/DNA hybrid in an isolated form, suggesting that this domain is involved in substrate binding. SIB1 RBD-RNase HI exhibited the enzymatic activity both in vitro and in vivo. Its optimum pH and metal ion requirement were similar to those of SIB1 RNase HI, E. coli RNase HI, and human RNase H1. The specific activity of SIB1 RBD-RNase HI was comparable to that of E. coli RNase HI and was much higher than those of SIB1 RNase HI and human RNase H1. SIB1 RBD-RNase HI showed poor cleavage-site specificity for oligomeric substrates. SIB1 RBD-RNase HI was less stable than E. coli RNase HI but was as stable as human RNase H1. Database searches indicate that several bacteria and archaea contain an RBD-RNase HI. This is the first report on the biochemical characterization of RBD-RNase HI.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo.

A DNA fragment encoding Ribonuclease H (EC 3. 1.26.4) was isolated from an extreme thermophilic bacterium, Thermus thermophilus HB8, by its ability to complement the temperature-sensitive growth of an Escherichia coli rnhA deficient mutant. The primary amino acid sequence showed 56% similarity to that of E. coli RNase HI but little or no homology to E. coli RNase HII. Enzymes derived from thermophilic organisms tend to have fewer cysteines than their bacterial counterparts. However, T. thermophilus RNase H has one more cysteine than its E. coli homologue. Stability of the RNase H in extracts of T. thermophilus to elevated temperatures was the same for the protein expressed in E. coli. T. thermophilus RNase H should, therefore, be a useful tool for editing RNA-DNA hybrid molecules at higher temperatures and may also be stable enough to be used in a cyclical process. It was suggested that regulation of expression of the RNase H may be different from that of E. coli. RNase HI.     

小花棘豆(Oxytripis glabra DC)毒素基因的cDNA克隆及序列测定

在完成小花棘豆毒素 95 %氨基酸序列的基础上 ,根椐已知的氨基酸序列 ,设计合成了特异简并引物 .以小花棘豆总RNA为模板 ,逆转录合成cDNA第一链 ,用置换法合成双链cDNA .用特异引物对此双链cDNA进行PCR扩增 ,将扩增后的目的基因与用SmaⅠ酶切的质粒pUC 18连接 ,转化大肠杆菌JM10 7.筛选阳性克隆进行序列分析 ,获得了OXY基因的全部序列 .经测序后测得基因序列与原氨基酸序列对照完全一致 .GenBnak数据检索说明 ,OXY基因编码序列确定是一个从未报道的序列 .此研究结果对该毒素的应用研究奠定了基础 .     

The SCO2299 gene from Streptomyces coelicolor A3(2) encodes a bifunctional enzyme consisting of an RNase H domain and an acid phosphatase domain

The SCO2299 gene from Streptomyces coelicolor encodes a single peptide consisting of 497 amino acid residues. Its N-terminal region shows high amino acid sequence similarity to RNase HI, whereas its C-terminal region bears similarity to the CobC protein, which is involved in the synthesis of cobalamin. The SCO2299 gene suppressed a temperature-sensitive growth defect of an Escherichia coli RNase H-deficient strain, and the recombinant SCO2299 protein cleaved an RNA strand of RNA.DNA hybrid in vitro. The N-terminal domain of the SCO2299 protein, when overproduced independently, exhibited RNase H activity at a similar level to the full length protein. On the other hand, the C-terminal domain showed no CobC-like activity but an acid phosphatase activity. The full length protein also exhibited acid phosphatase activity at almost the same level as the C-terminal domain alone. These results indicate that RNase H and acid phosphatase activities of the full length SCO2299 protein depend on its N-terminal and C-terminal domains, respectively. The physiological functions of the SCO2299 gene and the relation between RNase H and acid phosphatase remain to be determined. However, the bifunctional enzyme examined here is a novel style in the Type 1 RNase H family. Additionally, S. coelicolor is the first example of an organism whose genome contains three active RNase H genes.     

Nucleotide sequences required for a ColE1-type plasmid to replicate in Escherichia coli cells with or without RNase H

To elucidate the replication mechanism of a ColE1-type plasmid in RNase H-deficient (rnh-) strains of Escherichia coli, we constructed plasmid derivatives that deleted the whole, or a part, of the 5'-AAAAA-3' sequence (positions -3 to +2) that acts as the origin of replication in vivo and in vitro in the presence of RNase H. The activity of plasmid replication in rnh+ cells was found to be reduced by alterations of the AAAAA sequence. The activity could be restored when the derivatives, retaining the upstream sequence down to -8, regained a sequence containing at least two A residues in the region from -3 to +2. By contrast, replication in rnh- cells was maintained at high levels even when the deletion included the AAAAA sequence and extended up to position -7. The activity in rnh- cells decreased as deletions proceeded to -8 and further up to -17, and was abolished completely by further upward deletions. We concluded that in rnh- cells the plasmid replicates by a mechanism that operates only when RNase H is inactive. This RNase H-sensitive replication in rnh- cells seems to require the RNA-DNA hybrid formation that is also required for RNase H-dependent replication in rnh+ cells. The hybrid formation probably contributes by unwinding a portion of DNA from which replication can be initiated.     

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.     

Studies on a Vibrio vulnificus Functional Ortholog of Escherichia coli RNase E Imply a Conserved Function of RNase E-like Enzymes in Bacteria

RNase E (Rne) plays a key role in the processing and degradation of RNA in Escherichia coli. In the genome of Vibrio vulnificus, one open reading frame potentially encodes a protein homologous to E. coli RNase E, designated RNase EV, which N-terminal (1-500 amino acids) has 86.4% amino acid identity to the N-terminal catalytic part of RNase E (N-Rne). Here, we report that both the full-length and the N-terminal part of RNase EV (N-RneV) functionally complement E. coli RNase E and their expression consequently supports normal growth of RNase E-depleted E. coli cells. E. coli cells expressing N-RneV showed copy numbers of ColE1-type plasmid similar to that of E. coli cells expressing N-Rne, indicating in vivo ribonucleolytic activity of N-RneV on RNA I, an antisense regulator of ColE1-type plasmid replication. In vitro cleavage assays further showed that N-RneV has cleavage activity and specificity of RNase E on RNase E-targeted sequence of RNA I (BR13). Our findings suggest that RNase E-like proteins have conserved enzymatic properties that determine substrate specificity across species.     

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were approximately 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by approximately 0.2 M and approximately 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.     

Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene.

The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].     

Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.     

Expression of RNase Rh from Rhizopus niveus in yeast and characterization of the secreted proteins.

The full-length cDNA encoding RNase Rh, which is secreted extracellularly by Rhizopus niveus, was isolated and its nucleotide sequence was determined. It was placed under control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene of Saccharomyces cerevisiae in a high expression vector in yeast. Since yeast cells transformed by this plasmid poorly secreted RNase into the medium, the plasmid pYE RNAP-Rh was constructed, in which the signal sequence of RNase Rh was replaced by the prepro-sequence of aspartic proteinase-I, one of the extracellular enzymes secreted by R. niveus. Yeast cells harboring pYE RNAP-Rh produced RNase efficiently (ca. 40 micrograms/ml) into the medium. The product was a mixture of six enzymes (RNase RNAP-Rhs) having 3, 5, 9, 13, 14, and 16 additional amino acid residues attached to the amino terminus of the mature RNase Rh. The major product was the RNase with three additional amino acids at the amino terminus. Limited digestion of RNase RNAP-Rhs with staphylococcal V8 protease succeeded in shortening the various lengths of extra amino acid residues attached to the amino terminus of RNase Rh, yielding an RNase that has 3 additional amino acids at the amino terminus. It has been named RNase RNAP-Rh. The RNase RNAP-Rh showed the same specific activity and CD spectra as those of RNase Rh, suggesting that the two have similar conformations to each other around aromatic amino acid residues and the peptide backbone.