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PubMed1989

An immunological determinant of RNase P protein is conserved between Escherichia coli and humans.

RNase P, an enzyme with RNA and protein subunits, cleaves tRNA precursor molecules to form the 5' termini of mature tRNAs in both prokaryotes and eukaryotes. Rabbit antibodies made against the protein subunit, C5 protein, of Escherichia coli RNase P bound RNase P protein from E. coli and Bacillus subtilis in immunoblots and solid-phase immunoassays. These rabbit anti-C5 antibodies also bound a protein (Mr approximately 40,000) in preparations of RNase P from human (HeLa) cells and depleted the enzymatic activity from preparations of RNase P from both human and E. coli cells. Finally, rabbit anti-C5 antibodies immunoprecipitated from crude extracts of human cells a ribonucleoprotein complex containing H1 RNA, the putative RNA component of human RNase P. These results show that an antigenic determinant is shared by C5 protein from E. coli RNase P and a protein component of RNase P from human cells.

Antibodies↗
PubMed1993

Functional interaction of heat shock protein GroEL with an RNase E-like activity in Escherichia coli.

The highly specific endoribonuclease activities of RNase E (which processes ribosomal 9S RNA into p5S RNA) and RNase K (which initiates decay of the ompA mRNA) are inferred to play a central role in RNA processing and mRNA decay in Escherichia coli. In vivo both activities are affected by a conditional mutation of the ams/rne gene that seems to be complemented at nonpermissive temperatures by a fragment of the groEL gene. Analysis of the relationship between the two nucleases and the heat shock protein revealed that GroEL interacts functionally with an RNase E-like activity but not with an RNase K activity, a groEL mutation affected 9S RNA processing but not ompA mRNA cleavage, RNase E activity could be precipitated with an antibody against GroEL, and a highly purified GroEL preparation contained RNase E activity but not RNase K activity. When purifying RNase E activity, we obtained a preparation containing two major proteins of 60 and 17 kDa. The size and the N-terminal sequence identified the 60-kDa protein as GroEL.

Amino Acid Sequence↗
PubMed1999

RNase P RNAs from some Archaea are catalytically active.

The RNA subunits of RNase Ps of Archaea and eukaryotes have been thought to depend fundamentally on protein for activity, unlike those of Bacteria that are capable of efficient catalysis in the absence of protein. Although the eukaryotic RNase P RNAs are quite different than those of Bacteria in both sequence and structure, the archaeal RNAs generally contain the sequences and structures of the bacterial, phylogenetically conserved catalytic core. A spectrum of archaeal RNase P RNAs were therefore tested for activity in a wide range of conditions. Many remain inactive in ionically extreme conditions, but catalytic activity could be detected from those of the methanobacteria, thermococci, and halobacteria. Chimeric holoenzymes, reconstituted from the Methanobacterium RNase P RNA and the Bacillus subtilis RNase P protein subunits, were functional at low ionic strength. The properties of the archaeal RNase P RNAs (high ionic-strength requirement, low affinity for substrate, and catalytic reconstitution by bacterial RNase P protein) are similar to synthetic RNase P RNAs that contain all of the catalytic core of the bacterial RNA but lack phylogenetically variable, stabilizing elements.

Archaea↗
PubMed1996

RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme.

Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.

Base Sequence↗
PubMed1997

Mutations within the primer grip region of HIV-1 reverse transcriptase result in loss of RNase H function.

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3'-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5'-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5'-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3'-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5'-RNA-directed RNase H catalysis.

Binding Sites↗
PubMed1997

Processing of the rne transcript by an RNase E-independent amino acid-dependent mechanism.

RNase E is encoded by the rne (also known as ams or hmp) gene and is the principal enzyme that controls the chemical decay of bulk mRNA in Escherichia coli. Earlier work has shown that RNase E degrades its own mRNA, autoregulating production of the RNase E protein. Here we show that in cells lacking RNase E activity, the 3.6-kilobase rne gene transcript is cleaved site specifically at two locations near its center by a novel endonuclease whose activity is modulated by the presence or absence of amino acids in the culture medium. These cleavages produce a 2-kilobase RNase E-sensitive RNA fragment corresponding to the 3' half of the transcript. Using primer extension and RNase protection analysis, we mapped RNase E-independent cleavages to sites 1558 and 1576 nucleotides from the 5' end of the rne transcript (coordinates 1738 and 1747 of the rne gene). Our results indicate the existence of a previously unknown RNase E-independent mechanism for degradation of rne transcripts and further demonstrate that this mechanism responds to changes in cell growth conditions.

Amino Acids↗
PubMed1998

Bacillus subtilis RNase III cleaves both 5'- and 3'-sites of the small cytoplasmic RNA precursor.

Bacillus subtilis small cytoplasmic RNA (scRNA) is a member of the signal recognition particle RNA family. It is transcribed as a 354-nucleotide primary transcript and processed to a 271-nucleotide mature scRNA. In the precursor, the 5'- and 3'-flanking regions form a stable double-stranded structure based on their complementary sequence. This structure is similar to those of substrates for the double-stranded RNA processing enzyme, RNase III. The B. subtilis enzyme that has similar activity to Escherichia coli RNase III has been purified and is designated Bs-RNase III. Recently, B. subtilis rncS has been shown to encode Bs-RNase III (Wang, W., and Bechhofer, D. H. (1997) J. Bacteriol. 179, 7379-7385). We show here that Bs-RNase III and the purified His-tagged product of rncS cleave pre-scRNA at both 5'- and 3'-sites to produce an intermediate scRNA (scRNA-275), although processing at the 3'-site is less efficient. The 5'-end of scRNA-275 was identical to that of the mature scRNA, whereas it contains four excess nucleotides at the 3'-end. Bs-RNase III cleavage yields a two-base 3'-overhang, which is consistent with the manner in which E. coli RNase III cleaves. We also show that truncation of the rncS gene affected processing, and significant amounts of an intermediate scRNA (scRNA-275) were found to accumulate in the rncS-truncated mutant. It is concluded that Bs-RNase III is an enzyme that processes pre-scRNA.

Bacillus subtilis↗
PubMed1998

Activation/attenuation model for RNase H. A one-metal mechanism with second-metal inhibition.

Ribonucleases H (RNases H) comprise a family of metal-dependent enzymes that catalyze the hydrolysis of the 3'-O---P bond of RNA in RNA.DNA hybrids. The mechanism by which RNases H use active-site metal(s) for catalysis is unclear. Based upon the seemingly contradictory structural observations of one divalent metal bound to Escherichia coli RNase HI and two divalent metals bound to the HIV RNase H domain, two models explaining RNase H metal dependence have been proposed: a one-metal mechanism and a two-metal mechanism. In this paper, we show that the Mn2+-dependent activity of E. coli RNase HI is not consistent with either of these mechanisms. RNase H activity in the presence of Mn2+ is complex, with activation and inhibition of the enzyme at low and high Mn2+ concentrations, respectively. Mutations at Asp-134 result in a partial loss of this inhibition, with little effect on activation. Neutralization of His-124 by mutation to Ala results in an enzyme with a significantly decreased specific activity and an absolute loss of Mn2+ inhibition. Inhibition by high Mn2+ concentrations is shown to be due to a reduction in kcat; this attenuation has a critical dependence on the presence of His-124. Based upon these results, we propose an "activation/attenuation" model explaining the metal dependence of RNase H activity where one metal is required for enzyme activation and binding of a second metal is inhibitory.

Amino Acid Substitution↗
PubMed1998

Identification of a potent DNase activity associated with RNase T of Escherichia coli.

RNase T was first identified as an enzyme responsible for end turnover of tRNA in Escherichia coli. Its activity, specific for tRNA-C-C-A, catalyzes the release of tRNA-C-C and AMP. RNase T, along with several other RNases, plays a role in maturation of several other RNA species by a similar limited nuclease activity. In previous work, we identified the gene for RNase T, rnt, as a high copy suppressor of the UV sensitivity conferred by deficiency in three single-strand DNA-specific exonucleases, RecJ, exonuclease I, and exonuclease VII. This suggested that RNase T may process DNA substrates as well. In this work, we show that purified RNase T possesses a potent 3' to 5' single-strand DNA-specific exonucleolytic activity. Its Km for single-strand DNA substrates is many orders of magnitude lower than that for tRNA, suggesting that single-strand DNA may be a natural biological substrate for RNase T. We suggest that the DNase activity of RNase T may play a role in end trimming reactions during DNA recombination and/or DNA repair.

DNA Repair↗
PubMed2000

Human RNase III is a 160-kDa protein involved in preribosomal RNA processing.

A human RNase III gene encodes a protein of 160 kDa with multiple domains, a proline-rich, a serine- and arginine-rich, and an RNase III domain. The expressed purified RNase III domain cleaves double-strand RNA and does not cleave single-strand RNA. The gene is ubiquitously expressed in human tissues and cell lines, and the protein is localized in the nucleus of the cell. The levels of transcription and translation of the protein do not change during different phases of the cell cycle. However, a significant fraction of the protein in the nucleus is translocated to the nucleolus during the S phase of the cell cycle. That this human RNase III is involved in processing of pre-rRNA, but might cleave at sites different from those described for yeast RNase III, is shown by antisense inhibition of RNase III expression. Inhibition of human RNase III expression causes cell death, suggesting an essential role for human RNase III in the cell. The antisense inhibition technique used in this study provides an effective method for functional analysis of newly identified human genes.

Amino Acid Sequence↗
PubMed2000

Co-crystal of Escherichia coli RNase HI with Mn2+ ions reveals two divalent metals bound in the active site.

Ribonuclease H (RNase H) selectively degrades the RNA strand of RNA.DNA hybrids in a divalent cation-dependent manner. Previous structural studies revealed a single Mg(2+) ion-binding site in Escherichia coli RNase HI. In the crystal structure of the related RNase H domain of human immunodeficiency virus reverse transcriptase, however, two Mn(2+) ions were observed suggesting a different mode of metal binding. E. coli RNase HI shows catalytic activity in the presence of Mg(2+) or Mn(2+) ions, but these two metals show strikingly different optimal concentrations. Mg(2+) ions are required in millimolar concentrations, but Mn(2+) ions are only required in micromolar quantities. Based upon the metal dependence of E. coli RNase HI activity, we proposed an activation/attenuation model in which one metal is required for catalysis, and binding of a second metal is inhibitory. We have now solved the co-crystal structure of E. coli RNase HI with Mn(2+) ions at 1.9-A resolution. Two octahedrally coordinated Mn(2+) ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.

Aspartic Acid↗
PubMed2002

The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.

The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of the Trypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His(6)-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [(3)H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [(32)P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg(2+)-dependent, and it is also active in the presence of the Mn(2+) ion. The optimal condition of RNase H activity is found at pH 8 and 37 degrees C, although it also has significant enzymatic activity at 19 degrees C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.

Amino Acid Sequence↗
PubMed2002

RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin.

We analyzed healthy human skin for the presence of endogenous antimicrobial proteins that might explain the unusually high resistance of human skin against infections. A novel 14.5-kDa antimicrobial ribonuclease, termed RNase 7, was isolated from skin-derived stratum corneum. RNase 7 exhibited potent ribonuclease activity and thus may contribute to the well known ribonuclease activity of human skin. RNase 7 revealed broad spectrum antimicrobial activity against many pathogenic microorganisms and remarkably potent activity (lethal dose of 90% < 30 nm) against a vancomycin-resistant Enterococcus faecium. Molecular cloning from skin-derived primary keratinocytes and purification of RNase 7 from supernatants of cultured primary keratinocytes indicate that keratinocytes represent the major cellular source in skin and that RNase 7 is secreted. RNase 7 mRNA expression was detected in various epithelial tissues including skin, respiratory tract, genitourinary tract, and at a low level, in the gut. In addition to a constitutive expression, RNase 7 mRNA was induced in cultured primary keratinocytes by interleukin-1beta, interferon-gamma, and bacterial challenge. This is the first report demonstrating RNases as a novel class of epithelial inducible antimicrobial proteins, which may play an important role in the innate immune defense system of human epithelia.

Amino Acid Sequence↗
PubMed2002

Mechanism of action of RNase T. II. A structural and functional model of the enzyme.

A detailed structural and functional model of E. coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation. In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified. In the model, these segments cluster to form a positively charged surface patch. However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center. We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site. In support of this model, mutagenetic studies show that one NBS1 residue, Arg(13), sits at the catalytic center despite being on the opposite side of the monomer. Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding. These data explain why RNase T must dimerize to function. The model provides a detailed framework on which to explain the mechanism of action of RNase T.

Amino Acid Motifs↗
PubMed2002

Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines.

Human RNase H1 is active only under reduced conditions. Oxidation as well as N-ethylmaleimide (NEM) treatment of human RNase H1 ablates the cleavage activity. The oxidized and NEM alkylated forms of human RNase H1 exhibited binding affinities for the heteroduplex substrate comparable with the reduced form of the enzyme. Mutants of human RNase H1 in which the cysteines were either deleted or substituted with alanine exhibited cleavage rates comparable with the reduced form of the enzyme, suggesting that the cysteine residues were not required for catalysis. The cysteine residues responsible for the observed redox-dependent activity of human RNase H1 were determined by site-directed mutagenesis to involve Cys(147) and Cys(148). The redox states of the Cys(147) and Cys(148) residues were determined by digesting the reduced, oxidized, and NEM-treated forms of human RNase H1 with trypsin and analyzing the cysteine containing tryptic fragments by micro high performance liquid chromatography-electrospray ionization-Fourier transform ion cyclotron mass spectrometry. The tryptic fragment Asp(131)-Arg(153) containing Cys(147) and Cys(148) was identified. The mass spectra for the Asp(131)-Arg(153) peptides from the oxidized and reduced forms of human RNase H1 in the presence and absence of NEM showed peptide masses consistent with the formation of a disulfide bond between Cys(147) and Cys(148). These data show that the formation of a disulfide bond between adjacent Cys(147) and Cys(148) residues results in an inactive enzyme conformation and provides further insights into the interaction between human RNase H1 and the heteroduplex substrate.

Amino Acid Sequence↗
PubMed2006

Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus.

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.

Amino Acid Sequence↗
PubMed2006

Substrate recognition and catalysis by the exoribonuclease RNase R.

RNase R is a processive, 3' to 5' hydrolytic exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3' overhang is present. Using a variety of specifically designed substrates, we show here that a 3' overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5' overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3' to 5' polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3' termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.

Catalysis↗
PubMed1996

An RNase P RNA subunit mutation affects ribosomal RNA processing.

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

Base Sequence↗