Regiospecific cleavage of RNA by RNase H from E. coli in the presence of a complementary oligonucleotide with inserted alternating 2'-O-methylcytidine residues

1990 ◽  
Vol 55 (11) ◽  
pp. 2781-2786 ◽  
Author(s):  
Valerii G. Metelev ◽  
Natalya F. Krynetskaya ◽  
Andrei A. Purmal ◽  
Zoya A. Shabarova ◽  
Zdeněk Točík ◽  
...  
Keyword(s):  
Rnase H ◽  
5S Rrna ◽  
E Coli ◽  
Complementary Oligonucleotide

Undecanucleotide dA-dC-dC-dA-Cm-Cm-dG-Cm-dG-Cm-dT (Cm = 2'-O-methylcytidine) was prepared and used as a probe for splitting of 5S rRNA fragment by RNase H (E. coli). It was found that substitution of 2'-deoxycytidine of d(ACCACCGCGCT) by 2'-O-methylcytidine in positions 5, 6, 8 and 10 provided regiospecific splitting of RNA fragment by RNase H in the presence of modified probe between U25 and C26.

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

scholarly journals A small nuclear RNA, U5, can transform cells in vitro.

1989 ◽  
Vol 9 (10) ◽  
pp. 4345-4356 ◽  
Author(s):  
K Hamada ◽  
T Kumazaki ◽  
K Mizuno ◽  
K Yokoro
Keyword(s):  
Cell Transformation ◽  
Rnase H ◽  
Low Molecular Weight ◽  
Small Nuclear Rna ◽  
Fibroblastic Cell ◽  
Myc Oncogene ◽  
Chemically Induced ◽  
Complementary Oligonucleotide ◽  
Nuclear Rna

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.

scholarly journals Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

1988 ◽  
Vol 8 (2) ◽  
pp. 814-821 ◽  
Author(s):  
M Zillmann ◽  
M L Zapp ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Splice Junction ◽  
Rnase H ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
Complementary Oligonucleotide ◽  
Exon 1 ◽  
Precursor Rna ◽  
Splice Junctions

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

scholarly journals Evidence from molecular dynamics simulations of conformational preorganization in the ribonuclease H active site

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 67 ◽  
Author(s):  
Kate A. Stafford ◽  
Arthur G. Palmer III
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Active Site ◽  
Extensive Study ◽  
Rnase H ◽  
Dynamic Feature ◽  
Magnesium Ions ◽  
E Coli ◽  
Domains Of Life ◽  
Dynamics Simulations

Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold.

Three-Dimensional Image Reconstruction of the 50S Subunit from Escherichia Coli Ribosomes Lacking 5S-rRNA

1990 ◽  
Vol 48 (1) ◽  
pp. 284-285
Author(s):  
Michael Radermacher ◽  
Volker Nowotny ◽  
Robert Grassucci ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
5S Rrna ◽  
Scattering Data ◽  
23S Rrna ◽  
Reconstruction Technique ◽  
Electron Dose ◽  
E Coli ◽  
Phenol Extraction ◽  
Negative Stain

In earlier studies the structure of the 50S ribosomal subunit from E. coli has been determined from electron micrographs, using the single exposure random conical reconstruction technique. For the understanding of the function of ribosomes the single proteins and ribosomal RNAs need to be located within the ribosome structure. For localization of most proteins immunoelectron microscopy and neutron scattering data are available. The current study’s goal is the localization of the 5S rRNA from a comparison of the structures of complete 50S subunits with that of subunits reconstituted omitting the 5S rRNA.By Phenol extraction of purified 50S subunits the rRNA fraction was separated form the total protein fraction (TP50). This rRNA fraction was separated into 23S rRNA and 5S rRNA via HPLC on a DEAE-column. The total reconstitution of 23S rRNA and the equivalent amount of TP50 resulted in particles lacking the 5S rRNA. For electron microscopy the subunits were prepared in a negative stain sandwich preparation.Twenty tilt pairs at 50° tilt and 0° were recorded, with an electron dose of approximately 10el/A2 and a magnification of 49,000. A total of 983 particles were selected from these data. The 0° images were aligned using the procedure described in.

A small nuclear RNA, U5, can transform cells in vitro

1989 ◽  
Vol 9 (10) ◽  
pp. 4345-4356
Author(s):  
K Hamada ◽  
T Kumazaki ◽  
K Mizuno ◽  
K Yokoro
Keyword(s):  
Cell Transformation ◽  
Rnase H ◽  
Low Molecular Weight ◽  
Small Nuclear Rna ◽  
Fibroblastic Cell ◽  
Myc Oncogene ◽  
Chemically Induced ◽  
Complementary Oligonucleotide ◽  
Nuclear Rna

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.

scholarly journals Initial analysis of 750 MHz NMR spectra of selectively 15 N-G,U labelled E. coli 5S rRNA

FEBS Letters ◽  
1996 ◽  
Vol 385 (1-2) ◽  
pp. 114-118 ◽  
Author(s):  
Matthias Grüne ◽  
Matthias Görlach ◽  
Vukic Soskic ◽  
Sven Klussmann ◽  
Rolf Bald ◽  
...  
Keyword(s):  
Nmr Spectra ◽  
5S Rrna ◽  
E Coli ◽  
Initial Analysis

scholarly journals Tracing a protein’s folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Shion An Lim ◽  
Eric Richard Bolin ◽  
Susan Marqusee
Keyword(s):  
Hydrogen Exchange ◽  
Rnase H ◽  
Ancestral Sequence ◽  
Folding Pathway ◽  
Folding Intermediate ◽  
Evolutionary Time ◽  
E Coli ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction

The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a protein’s folding pathway.

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