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PubMed1991

Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity.

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.

Amino Acid Sequence↗
PubMed2004

Comparison of the effects of RNase-negative and wild-type classical swine fever virus on peripheral blood cells of infected pigs.

Elimination of the RNase activity of classical swine fever virus (CSFV) glycoprotein E(rns) was previously shown to result in virus attenuation. Specific reduction of B cell numbers in the peripheral blood, a typical symptom of CSFV infection in pigs, was not detected on infection with the RNase-negative mutant C-H346Delta [Meyers et al. (1999). J Virol 73, 10224-10235]. The present report shows that this feature is restricted to this specific virus mutant, and does not represent a general property of RNase-negative CSFV. The effects induced by infection with two other RNase-negative and wild-type (wt) CSFV strains on the composition of peripheral blood cells have been further analysed. For all viruses, not only general leukopenia but also a reduction of different subsets of leukocytes (T-lymphocytes, monocytes and granulocytes) was detected. Similar to the results with B-cells, no significant differences with regard to changes in cell number were determined for RNase-negative mutants and wt virus during the initial phase of infection. Later, the values returned to pre-infection levels for the mutants, but stayed at low levels in the wt virus-infected animals. A major difference was reflected in the virus load of the infected animals, which was dramatically higher for pigs infected with wt CSFV, so that reduction of the virus load represents a further marker for attenuation resulting from RNase destruction. Attenuation was also detectable for the RNase-negative mutant C-W300G, which showed rapid reversion to the wt sequence within the infected pig. The prevention of fatal disease after infection with C-W300G is apparently determined during the short time between infection and reversion, as the virus revertant reisolated from infected pigs was shown to be virulent when used for infection in a follow-up study. Reversion of C-W300G was also detected in tissue culture during passage on swine testis epithelioid cells and porcine transformed kidney (MAX) cells, whereas the mutation was stable when SK6 or 38A1D cells were tested.

Amino Acid Substitution↗
PubMed2005

RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs.

Hfq-binding antisense small RNAs of Escherichia coli, SgrS and RyhB, mediate the destabilization of target mRNAs in an RNase E-dependent manner. SgrS, whose expression is induced in response to phosphosugar stress, act on the ptsG mRNA encoding a major glucose transporter, while RyhB, whose expression is induced in response to Fe depletion, acts on several mRNAs encoding Fe-binding proteins. In this report, we addressed the question of how SgrS and RyhB RNAs cooperate with RNase E to destabilize the target mRNAs. We demonstrate that Hfq along with SgrS and RyhB copurified with RNase E but not with truncated RNase E. In addition, we show that RNase E but not other degradosome components copurified with Hfq. Taken together, we conclude that RNase E forms variable ribonucleoprotein complexes with Hfq/small RNAs through its C-terminal scaffold region. These complexes, distinct from the RNA degradosome, may act as specialized RNA decay machines that initiate the degradation of mRNAs targeted by each small RNA. The present finding has uncovered the mechanical basis of mRNA destabilization mediated by bacterial small RNAs. The formation of ribonucleoprotein complexes containing RNases could be a general way by which small RNAs destabilize target mRNAs in both prokaryotes and eukaryotes.

Endoribonucleases↗
PubMed1992

Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site.

Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.

Base Composition↗
PubMed1994

Characterization of a unique protein component of yeast RNase MRP: an RNA-binding protein with a zinc-cluster domain.

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to cleave mitochondrial primer RNA sequences from a variety of sources. Most of the RNase MRP activity is found in the nucleus where it plays a role in the processing of 5.8S rRNA. A temperature-conditional point mutation in the yeast RNA component of the enzyme has been identified. This mutation results in a loss of normal rRNA processing at the nonpermissive temperature while cellular levels of the RNA component of RNase MRP remain stable. High-copy suppressor analysis of this point mutation was employed to identify interacting proteins. A unique suppressor, termed SNM1 (suppressor of nuclear mitochondrial endoribonuclease 1), was identified repeatedly. The SNM1 gene was localized to the right arm of chromosome IV, directly adjacent to the SNF1 gene, and it contains an open reading frame encoding a protein of 198 amino acids. The protein contains a leucine zipper motif, a zinc-cluster motif, and a serine/lysine-rich tail. The gene was found to be essential for viability in a yeast cell, consistent with it being a protein component of the RNase MRP ribonucleoprotein complex. Recombinant SNM1 protein binds RNA in both gel retardation and Northwestern assays. Antibodies raised against bacterially expressed proteins identified four separate species in yeast whole cell extracts. Antibodies directed against the SNM1 protein immunoprecipitated RNase MRP RNA from whole-cell extracts without precipitating the structurally and functionally related RNase P RNA. We propose that the SNM1 protein is an essential and specific component of the RNase MRP ribonucleoprotein complex, the first unique protein of this complex to be identified.

Amino Acid Sequence↗
PubMed1997

Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition.

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Journal Article↗
PubMed1991

Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE-dependent endonucleolytic processing.

Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript. Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism. The processing did not occur under non-permissive conditions in an E. coli rne mutant strain with a temperature-sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/allelic to rne. Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript. We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗
PubMed1993

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA.

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180 kDa. However, proteolytic fragments as small as 70 kDa, which can arise during purification, also exhibit RNase E activity. In vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cleaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.

Antibodies, Bacterial↗
PubMed2006

RNase R affects gene expression in stationary phase: regulation of ompA.

In nature, bacteria remain mostly in the stationary phase of the life cycle. Although mRNA is a major determinant of gene expression, little is known about mRNA decay in the stationary phase. The results presented herein demonstrate that RNase R is induced in stationary phase and is involved in the post-transcriptional regulation of ompA mRNA. This work is the first report of RNase R activity on a full length mRNA. In the absence of RNase R in a single rnr mutant, higher levels of ompA mRNA are found as a consequence of the stabilization of ompA full transcript. This effect is growth-phase-specific and not a growth-rate-dependent event. These higher levels of ompA mRNA were correlated with increases in the amounts of OmpA protein. We have also analysed the role of other factors that could affect ompA mRNA stability in stationary phase. RNase E was found to have the most important role, followed by polyadenylation. PNPase also affected the decay of the ompA transcript but RNase II did not seem to contribute much to this degradation process. The participation of RNase R in poly(A)-dependent pathways of decay in stationary phase of growth is discussed. The results show that RNase R can be a modulator of gene expression in stationary phase cells.

Bacterial Outer Membrane Proteins↗
PubMed1993

Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis.

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases. To examine this hypothesis we prepared the Ser57 --> Glu mutant of B. amyloliquefaciens, in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild-type RNases from B. amyloliquefaciens and close to that of RNases T1.

Bacillus↗
PubMed2005

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.

Acid Phosphatase↗
PubMed2006

The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis.

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2'-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA. Conformationally biased and flexible modified nucleotides were positioned at the junctions between the DNA and MOE residues of the chimeric substrates to modulate the effects of the MOE residues on human RNase H1 activity. The strong northern-biased locked-nucleic acid modification exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates. Enhanced cleavage rates were observed for the eastern-biased 2'-ara-fluorothymidine and bulge inducing N-methylthymidine modifications positioned at the 5'-DNA/3'-MOE junction as well as the southern-biased 2'-methylthiothymidine and conformationally flexible tetrafluoroindole (TFI) modifications positioned at the 5'-MOE/3'-DNA junction. The heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity, whereas nucleotide substitutions adjacent the TFI significantly affected the cleavage rate. Mismatch base pair(s) exhibited similar effects on human RNase H1 activity as the TFI modifications. The effects of the TFI modification and mismatch base pair(s) on human RNase H1 activity were influenced by the position of the modification relative to the nucleotides interacting with the catalytic site of the enzyme rather than the juxtaposition of the modification to the MOE residues. Finally, these results provide a method for enhancing the human RNase H1 activity of chimeric antisense oligonucleotides (ASO) as well as the design of more potent ASO drugs.

Base Sequence↗
PubMed2003

RNase L levels in peripheral blood mononuclear cells: 37-kilodalton/83-kilodalton isoform ratio is a potential test for chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a disorder characterized by debilitating fatigue associated with immunological abnormalities. The etiology remains unclear. A low-molecular-mass (37 kDa) isoform of RNase L has been described in peripheral blood mononuclear cell (PBMC) extracts, and the ratio of two isoforms of RNase L (37 kDa/83 kDa) has been proposed as a potential biochemical marker of CFS. In a prospective case-control study, we tested whether the RNase L 37-kDa/83-kDa ratio could discriminate a SFC population. We compared the ratio of RNase L isoforms in PBMCs from 11 patients with CFS (6 women and 5 men; mean age +/- standard deviation, 43.2 +/- 13.8 years) and PBMCs from 14 healthy well-matched volunteers (10 women and 4 men; age, 39.1 +/- 11.6 years). A ratio of RNase L of 0.4 used as a threshold allowed diagnosis of CFS with high sensitivity (91%; 95% confidence interval [CI], 57 to 99%) and specificity (71%; 95% CI, 41 to 90%). The positive and negative prognostic values were 71% (95% CI, 41 to 90%) and 91% (95% CI, 57 to 99%), respectively. In the absence of acute infection or chronic inflammation, a high RNase L ratio could distinguish CFS patients from healthy volunteers. Additional large studies and follow-up studies are required to confirm the stability of this high ratio of RNase L isoforms in a CFS group.

Adult↗
PubMed2006

Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression.

Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P(xyl)). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.

Bacillus subtilis↗
PubMed1992

RNase E-dependent cleavages in the 5' and 3' regions of the Escherichia coli unc mRNA.

The endonucleolytic processing of the unc mRNA encoding the eight subunits of the Escherichia coli F1F0-ATPase was studied. Northern (RNA) blots of mRNA expressed from a plasmid which contained the 3'-terminal portion of the operon including the uncDC sequences revealed, in addition to the expected 2-kb mRNA, a 0.5-kb RNA species which hybridized to an uncC antisense RNA probe. An uncD antisense RNA probe hybridized to only the 2-kb mRNA, implying that the upstream 1.5-kb fragment is rapidly degraded. The 5' end of the 0.5-kb fragment was determined by primer extension analysis to be 11 bases into the coding region of the uncC gene. In RNase E-deficient strains, the amount of the 0.5-kb product was strongly reduced while the levels of the precursor uncDC transcript remained high. Similar RNase E-dependent processing was found in the chromosomally encoded unc mRNA. As this RNase E-dependent cleavage directly inactivates uncC and appears to leave uncD susceptible to degradation, it seems unlikely to play a role in differential expression of the gene products but may be an important event in unc mRNA degradation. RNase E mutants also showed altered processing of the chromosomally encoded unc mRNA in the uncB region near the 5' end. The expected full-length (7-kb) transcript was recognized when RNA from the RNase E-deficient strain was subjected to Northern blot analysis with uncB- and uncC-specific probes. RNA from strains with functional RNase E lacked the 7-kb transcript but had a 6.2-kb mRNA detectable with the uncC but not the uncB probe. RNase E is therefore implicated in multiple cleavages of the unc mRNA.

Base Sequence↗
PubMed1993

Characterization of the RNase P RNA of Sulfolobus acidocaldarius.

RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.

Archaea↗
PubMed1999

Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168.

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.

Bacillus subtilis↗
PubMed2000

RNase III processing of intervening sequences found in helix 9 of 23S rRNA in the alpha subclass of Proteobacteria.

We provide experimental evidence for RNase III-dependent processing in helix 9 of the 23S rRNA as a general feature of many species in the alpha subclass of Proteobacteria (alpha-Proteobacteria). We investigated 12 Rhodobacter, Rhizobium, Sinorhizobium, Rhodopseudomonas, and Bartonella strains. The processed region is characterized by the presence of intervening sequences (IVSs). The 23S rDNA sequences between positions 109 and 205 (Escherichia coli numbering) were determined, and potential secondary structures are proposed. Comparison of the IVSs indicates very different evolutionary rates in some phylogenetic branches, lateral genetic transfer, and evolution by insertion and/or deletion. We show that the IVS processing in Rhodobacter capsulatus in vivo is RNase III-dependent and that RNase III cleaves additional sites in vitro. While all IVS-containing transcripts tested are processed in vitro by RNase III from R. capsulatus, E. coli RNase III recognizes only some of them as substrates and in these substrates frequently cleaves at different scissile bonds. These results demonstrate the different substrate specificities of the two enzymes. Although RNase III plays an important role in the rRNA, mRNA, and bacteriophage RNA maturation, its substrate specificity is still not well understood. Comparison of the IVSs of helix 9 does not hint at sequence motives involved in recognition but reveals that the "antideterminant" model, which represents the most recent attempt to explain the E. coli RNase III specificity in vitro, cannot be applied to substrates derived from alpha-Proteobacteria.

Alphaproteobacteria↗