scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase

1971 ◽  
Vol 121 (4) ◽  
pp. 621-627 ◽  
Author(s):  
B. Gregory Louis ◽  
P. S. Fitt
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Rna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Halophilic Bacteria ◽  
Nucleoside Triphosphate ◽  
Extreme Halophile ◽  
Univalent Cations ◽  
Absolute Requirement ◽  
Nucleoside Triphosphates

1. DNA-dependent RNA polymerase was purified 150-fold from crude extracts of the extreme halophile Halobacterium cutirubrum. 2. The enzyme requires the presence of native DNA and all four nucleoside triphosphates to incorporate 14C-labelled nucleoside triphosphate into an acid-insoluble ribonuclease-sensitive product. 3. It has an absolute requirement for both Mn2+ and Mg2+. 4. The polymerase requires a high salt concentration for stability, but is markedly inhibited by univalent cations. 5. Its molecular weight is very low compared with that of Escherichia coli RNA polymerase.

scholarly journals Intrasubunit nucleotide binding in ribonucleic acid polymerase

1978 ◽  
Vol 175 (1) ◽  
pp. 189-192 ◽  
Author(s):  
A D B Malcolm ◽  
J R Moffatt
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Periodate Oxidation ◽  
Nucleotide Binding ◽  
Alpha Subunit ◽  
Nucleoside Triphosphate ◽  
Competitive Inhibitors ◽  
Nucleoside Triphosphates ◽  
Derivatives Of

1. Periodate oxidation of the ribose ring was used to synthesize derivatives of nucleoside triphosphates. 2. These oxidized nucleoside triphosphates. 2. These oxidized nucleoside triphosphates are competitive inhibitors of RNA polymerase. 3. On incubation, together with NaBH4, these oxidized labelled nucleotides are covalently bound to Escherichia coli RNA polymerase. 4. Nucleoside triphosphate substrates decrease the extent of labelling. 5. A lysine residue in an alpha-subunit is labelled. 6. The significance of these results in relation to the location of the nucleotide-binding site is discussed.

scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum ribonucleic acid-dependent ribonucleic acid polymerase

1971 ◽  
Vol 121 (4) ◽  
pp. 629-633 ◽  
Author(s):  
B. Gregory Louis ◽  
P. S. Fitt
Keyword(s):  
Molecular Weight ◽  
Sodium Chloride ◽  
Nucleic Acid ◽  
Ribonucleic Acid ◽  
Halophilic Bacteria ◽  
Chloride Concentration ◽  
Nucleoside Triphosphate ◽  
Extreme Halophile ◽  
Sodium Chloride Concentration ◽  
The Stability

1. Crude extracts of the extreme halophile Halobacterium cutirubrum contain separable DNA-dependent and RNA-dependent RNA polymerases. 2. The RNA-dependent enzyme has been purified about 2800-fold. 3. It requires RNA, preferably of high molecular weight, and all four ribonucleoside triphosphates to incorporate 14C-labelled nucleoside triphosphate into an acid-insoluble, ribonuclease-sensitive product. 4. Both the stability and activity of the RNA polymerase are relatively insensitive to changes in potassium chloride or sodium chloride concentration, but incorporation is stimulated by both Mg2+ and Mn2+. 5. The molecular weight of the enzyme is about 17000–18000.

scholarly journals Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

1970 ◽  
Vol 117 (3) ◽  
pp. 623-631 ◽  
Author(s):  
Volker Neuhoff ◽  
Wolf-Bernhard Schill ◽  
Hans Sternbach
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Structure And Function ◽  
Disc Electrophoresis ◽  
Inhibitory Mechanism ◽  
And Function ◽  
Micro Analysis ◽  
Template Complex

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme–template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme–template complex to the same extent as by an RNA-synthesizing enzyme–template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.

scholarly journals A procedure for the rapid purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase (Short Communication)

1973 ◽  
Vol 133 (1) ◽  
pp. 201-203 ◽  
Author(s):  
Peter Humphries ◽  
David J. McConnell ◽  
Robert L. Gordon
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Short Communication ◽  
Gel Filtration ◽  
High Salt ◽  
Rapid Procedure ◽  
Complete Purification ◽  
Rapid Purification

A rapid procedure involving DNA–cellulose chromatography followed either by sedimentation in a high-salt glycerol gradient or by gel filtration is described for the complete purification of Escherichia coli DNA-dependent RNA polymerase.

scholarly journals Trigger loop folding determines transcription rate of Escherichia coli’s RNA polymerase

2014 ◽  
Vol 112 (3) ◽  
pp. 743-748 ◽  
Author(s):  
Yara X. Mejia ◽  
Evgeny Nudler ◽  
Carlos Bustamante
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Elongation Rate ◽  
Nucleoside Triphosphate ◽  
Transcription Rate ◽  
Nucleotide Analogs ◽  
Catalytic Center ◽  
Trigger Loop

Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP’s pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.

scholarly journals A semisynthetic organism engineered for the stable expansion of the genetic alphabet

2017 ◽  
Vol 114 (6) ◽  
pp. 1317-1322 ◽  
Author(s):  
Yorke Zhang ◽  
Brian M. Lamb ◽  
Aaron W. Feldman ◽  
Anne Xiaozhou Zhou ◽  
Thomas Lavergne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Base Pair ◽  
Genetic Information ◽  
Information Storage ◽  
Nucleoside Triphosphate ◽  
Proof Of Concept ◽  
Sequence Context ◽  
Form Of Life ◽  
Nucleoside Triphosphates ◽  
Genetic Alphabet

All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported thatEscherichia coligrown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.

Partial characterization of the mode of inhibition of Escherichia coli RNA polymerase by the mixed disulfide, CoASSG

1979 ◽  
Vol 57 (4) ◽  
pp. 336-345 ◽  
Author(s):  
William C. H. Bees ◽  
Peter C. Loewen
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Equilibrium Dialysis ◽  
Mixed Disulfide ◽  
Nucleoside Triphosphates ◽  
Noncompetitive Inhibitor ◽  
Fe Complex ◽  
Short Time ◽  
Dna Template

The coenzyme A – glutathione mixed disulfide (CoASSG), when complexed with iron, is capable of inhibiting the RNA polymerase of Escherichia coli. A modified procedure involving a short time of exposure to high salt allowed the reliable preparation of CoASSG–Fe which was active in inhibiting RNA polymerase. The CoASSG–Fe complex acted as a noncompetitive inhibitor for the incorporation of all four nucleoside triphosphates but had a greater effect on GMP and CMP incorporation than AMP and UMP incorporation. Neither temperature nor ionic-strength changes affected CoASSG–Fe inhibition, and the use of rifampicin showed that CoASSG–Fe did not inhibit either the initiation or elongation processes of the polymerase. CoASSG–Fe was a more effective inhibitor at low DNA-template concentrations and it was more effective in inhibiting the incorporation of CMP and GMP on simple dG-dC containing templates and the asymmetric polymer poly d(T-C)∙poly d(G-A). The inhibition of transcription of poly d(I-C) was less effective than the inhibition of transcription of poly d(G-C). Equilibrium dialysis in microdialysis cells showed that CoASSG–Fe could associate with DNA in the absence of RNA polymerase.

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