現(xiàn)代分子生物學(xué)之原核細(xì)胞轉(zhuǎn)錄學(xué)習(xí)教案

1、會計學(xué)1第一頁，共30頁。Principal Types of RNAs Produced in Cells: a particular segment of DNA is copied into RNA by the enzyme RNA polymerase: RNAP, RNA pol第1頁/共29頁第二頁，共30頁。General themes of transcription第2頁/共29頁第三頁，共30頁。Transcription selectively occurs at only certain parts of the genome, and makes a few to

2、thousands copies, transiently, variably in different cells, in different enviroment.第3頁/共29頁第四頁，共30頁。 RNA Polymerases第4頁/共29頁第五頁，共30頁。Mg 2+第5頁/共29頁第六頁，共30頁。第6頁/共29頁第七頁，共30頁。TerminationInitiationElongation第7頁/共29頁第八頁，共30頁。RNA polymerase binds at specific location: Promoter strength: 第8頁/共29頁第九頁，共30頁。

3、RNA Pol Leaves Its Footprint on a PromoterHow did we identify the promoter in DNA molecule?第9頁/共29頁第十頁，共30頁。In bacteria, its thefactor that makes RNA polymerase initiate transcription only at promotersC- terminal domainfactorHoloenzymeCore enzymeThefactor has four regions第10頁/共29頁第十一頁，共30頁。Two helic

4、es within region 4 form a for DNA binding 第11頁/共29頁第十二頁，共30頁。An recognizes and interact with bases on non-template strand at 10 through its aromatic AAsConformational change, spontaneously but irreversible, in the DNA-enzyme complex to a 第12頁/共29頁第十三頁，共30頁。RNA polymerase holoenzyme = + 第13頁/共29頁第十四頁

5、，共30頁。RNA polymerase holoenzyme = + + + + + + + + + + + +The region 3-4 linker of sigma factor (RNA mimic) lies in middle of the RNA exit channel in the open complex第14頁/共29頁第十五頁，共30頁。RNA polymerase holoenzyme = + 第15頁/共29頁第十六頁，共30頁。It opens DNA double helix between -11 and +3第16頁/共29頁第十七頁，共30頁。Init

6、ial transcription第17頁/共29頁第十八頁，共30頁。Initial transcriptionPromoter escape involves breaking polymerase-promoter and the polymerase core-sigma interactionshe region 3-4 linker of factor (RNA mimic): the RNAP repeatedly and short RNA products until they reach 10nt, otherwise polymerase is .第18頁/共29頁第十九

7、頁，共30頁。The polymerase remains stationary and DNA into itPolymerases active center relative to DNA template and synthesizes short transcripts before aborting-repeating until escaping the promoterEnergy stored in scrunching is released to promoter escape and dislodging of sigma factor第19頁/共29頁第二十頁，共30

8、頁。Transcription shifts into the elongation phase once a short stretch of RNA ( 10 nt) is synthesizedThe transition requires further conformational change in polymerase that leads it to grip the template more firmly第20頁/共29頁第二十一頁，共30頁。1. in the catalytic cleft2. (Only 8-9 nts remain base-paired with

9、the DNA template at any given time) 3. from the DNA template just behind the RNA pol, then the 第21頁/共29頁第二十二頁，共30頁。Although proofreading for RNA synthesis do exist, transcription() is less accurate than replication(); Pyrophosphorolytic editing: remove a correctly or incorrectly inserted ribonucleot

10、ide by reincorporation of PPi, but hover longer over mismatchHydrolytic editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. (stimulated by , which both enhance hydrolytic editing function and serve as elongation stimulating factors: ensure that polyme

11、rase elongates efficiently and help overcome “arrest” at the difficult regions)第22頁/共29頁第二十三頁，共30頁。The micrograph(under the electron microscope) shows many molecules of RNA polymerasesimultaneously transcribing each of two adjacent genes. Molecules of RNA polymerase are visible as a series of dots a

12、long the DNA with the newly synthesized transcripts (fine threads) attached to them.Multiple RNA pol molecules can transcribe the same gene at the same time, each following closely behind another; 第23頁/共29頁第二十四頁，共30頁。Terminators: the sequences that trigger RNA polymerase to dissociate from the DNARh

13、o-independent terminator : a short inverted repeat (20 bp) and a stretch of 8 A:T base pairs. 第24頁/共29頁第二十五頁，共30頁。Have less well-characterized RNA elements rut ( utilization)Rho binding can wrest the RNA from the polymerase-template complex using the energy from ATP hydrolysisRNA tread through the “Open ring”第25頁