Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new instruction theory for antibody formation is presented. The reverse flow of information from the amino-acid sequences of small antigenic determinants to an antideterminant RNA (aRNA) seems feasible. Prerequisites are specific activating enzymes, tRNAs, ATP as well as some kind of membrane assembling the anticodons of tRNAs linearly, analogous to the linear primary structure of stretched polypeptides. Once synthesized, aRNA might be replicated, utilized as transfer factor and transcribed by means of Reverse Transcriptase into aDNA. Further steps would be the fusion of this aDNA with genetical performed DNA-molecules already coding for the basic strucures of different classes of immunoglobulins by means of a terminal deoxynucleotidyl-transferase. This could be a chromosomal or extrachromosomal integration. The second hypothesis concerns antigen-induced immunosuppression and the phenomenon of nonresponsiveness (tolerance). An overwhelming proteolysis might give rise to a degradation of antigens or receptor templates for antigenic determinants located on the surface of macrophages. On later exposure to a similar antigen proteolytic enzymes are already preformed abolishing rapidly antigenic information. The third hypothesis concerns antibody-induced immunosuppression and tolerance. Antideterminant information is integrated into the genome or established extra-chromosomally. The continuous presence of antibodies sets in motion a sequence of reactions causing an accumulation of all information intermediates including a complementary DNA strand to the aRNA. On exposure to the corresponding antigen aRNA is transcribed. However, translation might be inhibited by hybridisation with the complementary aDNA strand as well as specific RNA hydrolysis by RNase H. Concerning the immunogenity of antibodies, a proteolytical mechanism might also be possible. Taking this into account a tolerance could be suspended in the following way: 1. by influencing the overwhelming proteolytical degradation of antigenic determinants with simultaneous antigenic stimulation; 2. by substitution of aRNA to induce blocked antibody synthesis.
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PMID:[A new instruction theory: possibility of a reverse flow of information from polypeptide sequences to RNA particularly in antibody synthesis, and the mechanisms of tolerance induction and immunosuppression (author's transl)]. 5 2

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.
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PMID:Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition. 14 Jan 66

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

The DNA replication system of bacteriophage T4 serves as a relatively simple model for the types of reactions and protein-protein interactions needed to carry out and coordinate the synthesis of the leading and lagging strands of a DNA replication fork. At least 10 phage-encoded proteins are required for this synthesis: T4 DNA polymerase, the genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, the genes 61, 41, and 59 primase-helicase, RNase H, and DNA ligase. Assembly of the polymerase and the accessory proteins on the primed template is a stepwise process that requires ATP hydrolysis and is strongly stimulated by 32 protein. The 41 protein helicase is essential to unwind the duplex ahead of polymerase on the leading strand, and to interact with the 61 protein to synthesize the RNA primers that initiate each discontinuous fragment on the lagging strand. An interaction between the 44/62 and 45 polymerase accessory proteins and the primase-helicase is required for primer synthesis on 32 protein-covered DNA. Thus it is possible that the signal for the initiation of a new fragment by the primase-helicase is the release of the polymerase accessory proteins from the completed adjacent fragment.
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PMID:Protein-protein interactions at a DNA replication fork: bacteriophage T4 as a model. 131 Sep 46

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.
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PMID:A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. 146 25

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.
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PMID:Alterations of RNase H sensitivity of the 3' splice site region during the in vitro splicing reaction. 165 Apr 57

Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports. This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract. Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract. LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs. RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP. LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP. LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49. Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation. These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA. Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site. These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway.
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PMID:The U2 small nuclear ribonucleoprotein particle associates with nuclear factors in a pre-mRNA independent reaction. 165 22

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.
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PMID:The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly. 185 Jan 27

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.
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PMID:Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. 240 71


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