Journal ArticleDOI
Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter.
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TLDR
High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter, finding that the dinucleotide always represents 50% of the total of all oligon nucleotides, even when conditions are manipulated to cause a 100-fold variation in this total.Abstract:
High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added Nevertheless, oligonucleotide synthesis persists under all conditions tested Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro Production of a long RNA transcript is then essentially an escape from this cycling reaction The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reactionread more
Citations
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Journal ArticleDOI
Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates
TL;DR: A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter to increase the variety of RNAs which can be made.
Book ChapterDOI
Synthesis of small RNAs using T7 RNA polymerase.
TL;DR: This chapter discusses how transcription templates are prepared by cleaving the plasmid DNA with a restriction enzyme of choice, and how nucleoside triphosphates are diluted to desired concentrations and are mixed in equimolar ratios to make a stock solution.
Journal ArticleDOI
Structural Basis of Transcription Initiation: RNA Polymerase Holoenzyme at 4 Å Resolution
TL;DR: The crystal structure of the initiating form of Thermus aquaticus RNA polymerase, containing coreRNA polymerase (α2ββ′ω) and the promoter specificity σ subunit, has been determined at 4 angstrom resolution.
Journal ArticleDOI
Function of a Bacterial Activator Protein that Binds to Transcriptional Enhancers
TL;DR: The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma factor (referred to as s Sigma 54-holoenzyme).
Journal ArticleDOI
Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching
Andrey Revyakin,Chenyu Liu,Chenyu Liu,Chenyu Liu,Richard H. Ebright,Terence R. Strick,Terence R. Strick +6 more
TL;DR: The results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter andRNAP-initiation-factor interactions in escape.
References
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Journal ArticleDOI
Regulatory Sequences Involved in the Promotion and Termination of RNA Transcription
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The use of thin acrylamide gels for DNA sequencing.
Frederick Sanger,Alan Coulson +1 more
TL;DR: Using the plus and minus [l] and the terminator [3] methods, serious variations in the distances between consecutive nucleotide bands in regions of dyad symmetry where base-paired loop structures can form are found, and this can lead to difficulties of interpretation.
Journal ArticleDOI
Mapping adenines, guanines, and pyrimidines in RNA
TL;DR: The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule and form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.