Transcription organic matter
RNA polymerase: a core comprising an enzyme and a factor σ
σ factor: in vitro transcription factor σ if not, does not have the specific transcription, RNA polymerase transcribes the double-stranded DNA all, but after the addition of factor σ, become specific transcription initiation.
Promoter: When we use with a gap of DNA transcription, RNA polymerase transcription can be normal, but obviously not specific, however, is not complete T4DNA have gaps, RNA polymerase difficult to encounter this natural gap, and therefore its transcriptional activity will be greatly decreased, if σ factor is present, it can be a normal RNA polymerase recognition and binding site of initiation of transcription, the polymerase binding site become promoter , thus guiding the role of factor σ polymerase specific promoter at the transcription start DNA.
RNA polymerase promoter binding:
We found that, RNA polymerase holoenzyme can be closely integrated with the DNA, and the core enzyme can only loosely bound, depending on whether they contain σ factor, combined with the whole enzyme half-life of 30-60h, in conjunction with the core enzyme half-life of only 1min, further experiments found that the combination of a tight site has eight, which is similar to the number of promoters, and the combination of loose location 1300, and appears in any position, so we can conclude: core-specific polymerase can not play beginning transcription DNA, because in fact there must be transcribed promoters involved. Continue found that when we raise the temperature, may enhance the binding tightness of the solution 25 ° off-rate was significantly higher than 37 ℃, because high temperature can promote the DNA melting, so we can conclude the following hypothesis: RNA polymerase in the first DNA binding loose while, until the discovery of the promoter, and the promoter of the holoenzyme complex termed loosely bound closure initiation complex , then, a portion of the holoenzyme such that unwinding of the DNA, when the composite is called open starting complex .
Promoter Structure: What kind of bacterial DNA structure in order to make RNA polymerase binds it? After David Pribnow compare a plurality of E.coli and phage promoters, we find a common area, with its center at the transcription start site about 10bp above the point at length about 6-7bp, we now call the -10 sequence or - block 10 ; thereafter, Mark Ptashne and colleagues found a short sequence, its center at a position upstream of the transcription start 35pb, thousands promoter analysis found, there is a consensus sequence for each block
TTGACa TAtAaT ------------------------------- -------------- --- ------------- -------------- ---------------------- AACTGt --------- ---------------- ATaTtA
Capital letters indicate the frequency of occurrence of this base in this position is relatively high, the probability of this makes it difficult to have a common sequence with exactly the same sequence of -10 and -35 sequences, however, as long as there exact match, it will be very active transcription in fact, the sequences do not match, the lower the promoter activity. In addition, the distance between the promoter elements is also important, -10 and -35 will be away from or close to that transcriptional activity decreased.
In addition -10 and -35 sequence (which we call a core promoter element) Some highly active promoter there is an additional element, called UP element , such as E.coli cells we 7 genes encoding rRNA of when the fast growth requires large rRNA, seven gene itself may direct transcription of a large amount, we consider a real UP element is a promoter, an RNA polymerase may be identified as UP element, and only in the presence of UP elements, transcription activity can be increased by 30 times. So when we can decide whether to enhance transcription of it? Indeed, the promoter further relates to three Fis sites between the -60 to -150, they Fis protein is a transcriptional activator binding site, they are not bound to RNA polymerase promoter is not called, but a class is called an enhancer transcriptional activation DNA element.
E.coli RNA gene promoter is further regulated by a pair of small molecules, both starting NTP (iNTP) and warning element guanosine diphosphate -3`- -5`- diphosphate (ppGpp), a large number of instructions when present intp high nucleotide concentrations.
Transcription initiation: