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To perform a run-off transcription assay, a gene of interest, including the promoter, is cloned into a plasmid. [4] The plasmid is digested at a known restriction enzyme cut site downstream from the transcription start site such that the expected mRNA run-off product would be easily separated by gel electrophoresis. [1] [2] [4]
Example of a T-REx system controlling the expression of shRNA. Tetracycline-controlled transcriptional activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).
Activator-binding sites may be located very close to the promoter or numerous base pairs away. [2] [3] If the regulatory sequence is located far away, the DNA will loop over itself (DNA looping) in order for the bound activator to interact with the transcription machinery at the promoter site. [2] [3]
The tac promoter consists of the '–35' region of the trp promoter and the '–10' region of the lac promoter (and differs from a related trc promoter by 1 bp [3]). The tac promoter is, therefore, inducible by IPTG (Isopropyl β- D -1-thiogalactopyranoside), whilst also allowing higher maximum gene expression than either the lac or trp promoters.
The Pribnow box (also known as the Pribnow-Schaller box) is a sequence of TATAAT of six nucleotides (thymine, adenine, thymine, etc.) that is an essential part of a promoter site on DNA for transcription to occur in bacteria.
The RNA polymerase core associates with the sigma factor to form RNA polymerase holoenzyme. Sigma factor reduces the affinity of RNA polymerase for nonspecific DNA while increasing specificity for promoters, allowing transcription to initiate at correct sites. The core enzyme of RNA polymerase has five subunits (protein subunits) (~400 kDa). [14]
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