RNA聚合酶识.ppt

1、,Chapter 5 Transcription,A. Transcription in prokaryotes,5.1 Basic principles of transcription,5.2 Escherichia coli RNA polymerase,5.3 The E. coli s70 promoter,5.4 transcription process.,An overview, the process of RNA synthesis ( initiation, elongation, termination),Properties, a subunit, b subunit

2、, b subunit, sigma (s) factor,Promoter, s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency,Promoter binding, unwinding, RNA chain initiation, elongation, termination (r factor),5.1: Basic principles of transcription,Transcription: an overview (comparison with replica

3、tion) The process of RNA synthesis: initiation, elongation, termination,5.1-1: Transcription: an overview,Key terms defined in this section (Gene VII),Coding strand of DNA has the same sequence as mRNA.Downstream identifies sequences proceeding further in the direction of expression; for example, th

4、e coding region is downstream of the initiation codon.,Gene X,Primary transcript,m7Gppp,AAAAAn,+1,upstream,downstream,mRNA,Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation c

5、odon is upstream of the coding region. Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene. Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription,RNA Terminator is a sequence of DNA, re

6、presented at the end of the transcript, that causes RNA polymerase to terminate transcription. RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases). Primary transcript is the original unmodified RNA product corresponding to a tran

7、scription unit.,Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule. 1 DNA molecule 2 DNA molecules Transcription: synthesis of one RNA molecule using one of the two DNA strands as a template. 1 DNA molecule 1 RNA molecule,Replicati

8、on-synthesis of the leading strand,the same direction as the replication fork moves,Replication- Synthesis of the Okazaki fragments,Opposite to the replication fork movement,Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli),Transcription,RNA synthesis occurs

9、in the 53 direction and its sequence corresponds to the sense strand (coding strand). The template of RNA synthesis is the antisense strand (template strand). Phosphodiester bonds: same as in DNA Necessary components: RNA polymerase, transcription factors, rNTPs, promoter mutations in rpoB affect al

10、l stages of transcription. Mutations in rpoC show that b also is involved at all stages.,b subunit may contain two domains responsible for transcription initiation and elongation Rifampicin (利福平)：has been shown to bind to the subunit, and inhibit transcription initiation by prokaryotic RNA pol. Muta

11、tion in rpoB gene can result in rifampicin resistance. Streptolydigins(利迪链菌素)：resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.,b subunit Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase Hyparin (肝素)：binds t

12、o the b subunit and inhibits transcription in vitro. Hyparin competes with DNA for binding to the polymerase. 2.b subunit may be responsible for binding to the template DNA .,1. 亚基由rpoB 基因编码； 2. RNA聚合酶的催化中心； Rifampicin (利福平)：与亚基结合可以抑制转录的起始，rpoB 基因突变导致对利福平的抗性； Streptolydigins(利迪链菌素)：抗性突变也定位于 rpoB 基因，

13、它抑制转录延伸，但不抑制起始； 3. 亚基可能含有两个结构域负责转录的起始和延伸；,大肠杆菌RNA聚合酶: b 亚基,1. b 亚基由rpoC 基因编码； 2. 与两个Zn2+ 离子结合参与RNA聚合酶的催化功能； Heparin (肝素)：在体外与 亚基结合并抑制转录； Heparin 与DNA竞争结合RNA聚合酶； 3. 亚基可能是负责与DNA模板的结合；,大肠杆菌RNA聚合酶: b亚基,5.2-5: Sigma (s) factor,Many prokaryotes contain multiple s factors to recognize different promoters.

14、The most common s factor in E. coli is s70. Binding of the s factor converts the core RNA pol into the holoenzyme. s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter s

15、 factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) Less amount of s factor is required in cells than that of the other subunits of the RNA pol.,1. 在启动子的识别上 因子至关重要，对于非特异性DNA位点核心酶的亲和力会降低（104），而对于相应的启动子亲和力会增加； 2. 当起始后(RNA chain is 8-9 nt) ， 因子会从RNA聚合酶中释放出来； 3. 在细胞中 因子的需要量比RNA聚合

16、酶其他亚基的需要量少；,大肠杆菌RNA聚合酶: s亚基,5.3: The E. coli s70 promoter,Promoter s70 size -10 sequence -35 sequence transcription start site promoter efficiency,5.3-1: Promoter,The specific short conserved DNA sequences: upstream from the transcribed sequence, and thus assigned a negative number (location) requir

17、ed for specific binding of RNA Pol. and transcription initiation (function) Were first characterized through mutations that enhance or diminish the rate of transcription of gene,Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.,5.

18、3-2,3,3. 当新生RNA链聚合1520bp时，构象进一步发生转变，形成延伸反应复合物，此时覆盖的长度为3040bp的DNA;,Footprinting is a technique derived from principles used in DNA sequencing. It is used to identify the specific DNA sequences that are bound by a particular protein.,RNA Polymerase Leaves Its FootPrint on a Promoter,Supplemental mater

19、ial,Footprinting,Footprinting,5.3-5: Transcription start site,Is a purine in 90% of all gene G is more common at position +1 than A There are usually a C and T on either side of the start nucleotide (i.e. CGT or CAT),The sequences of five E. coli promoters,K3-6: promoter efficiency,There is consider

20、able variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold .,The 35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase s factor. The -10 sequence is important for DNA unwinding. The sequen

21、ce around the start site influence initiation efficiency. The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength.,Some promoter sequence are not sufficien

22、tly similar to the consensus sequence to be strongly transcribed under normal condition, thus activating factor is required for efficient initiation. Example: Lac promoter P lac requires activating protein, cAMP receptor protein (CRP ), to bind to a site on the DNA close to the promoter sequence in

23、order to enhance polymerase binding and transcription initiation.,Weak promoters and activating factor,5.4 Transcription process,Promoter binding DNA unwinding RNA chain initiation RNA chain elongation RNA chain termination (r factor),Promoter binding,The searching process is extremely rapidly,Close

24、d complex: the initial complex of the polymerase with the base-paired promoter DNA),and 10 region,Link,The RNA polymerase core enyzme, a2bbw, has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable. The addition of s factor to the core enzyme markedly

25、 reduces the holoenzyme affinity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times. Overall, s factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site.,The role of s factor in promoter bi

26、nding,2. DNA unwinding,The initial unwinding of the DNA results in formation of an open complex with the polymerase, and this process is referred to as tight binding,+1,It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing and RNA synthesis. Negative su

27、percoiling enhances the transcription of many genes, since it facilitates unwinding. Some promoters are not. Exceptional example: promters for the enzyme subunits of DNA gyrase are inhibited by negative supercoiling, serving as an elegant feedback loop for DNA gyrase expression.,Negative supercoilin

28、g the second is called BRF (TFIIB-related factor); and the third is called B”.,TFIIIC: A and B boxes binding and a assembly factor to position TFIIIB,TFIIIB: DNA binding and RNA Pol III recruiting,5.7-3 5S rRNA genes,Tandemly arranged in a gene cluster. (In human, there is a single cluster of around

29、 2000 genes.) Transcription control regions (promoters) are organized similar to those of tRNA, except that C box is in place of B box. C box: +81-99 bp; A box: +50-65,3. Transcription factors: (1) The C box acts as the binding site for TFIIIA. (2) TFIIIA acts as an assembly factor which allows TFII

30、IC to interact with the 5S rRNA promoter. (3) The A box may also stabilize TFIIIC binding. (4) TFIIIC is then bound to DNA site near +1. (5) TFIIIB and TFIIIC interact to recruit RNA Pol III to initiate transcription.,TFIIIA,TFIIIC,TFIIIB,Pol III,5.7-4 Alternative RNA Pol III promoters,Many RNA Pol

31、III genes also rely on upstream sequences for regulation of their transcription e.g. U6 snRNA and Epstein-Barr virus Use only regulatory genes upstream from their transcription start sites.,U6 snRNA The coding region contains a characteristic A box that is not required for transcription. The upstrea

32、m sequence contains sequences typical of RNA Pol II promoters, including a TATA box at bases 30 to 23. Shares several other transcription factor binding sequences with many U RNA genes which are transcribed by RNA Pol II Suggestion: common transcription factors can regulate both RNA Pol II and Pol I

33、II genes,5.7-5 RNA Pol III termination,The RNA polymerase can terminate transcription without accessory factors. A cluster of A residue is often sufficient for termination. Xenopus borealis terminator: 5-GCAAAAGC-3,5.8 RNA Pol II genes: promoters and enhancers,RNA Pol II Cis-acting elements Promoter

34、s Upstream regulatory elements Enhancers,enhancer,Initiation element,Upstream element,TATA,Inr,mRNA,Down-stream element,5.8-1 RNA Pol II,located in nucleoplasm catalyzing the synthesis of the mRNA precursors for all protein-coding genes. RNA Pol -transcribed pre-mRNAs are processed through cap addit

35、ion, poly(A) tail addition and splicing.,5.8-2 Promoters,Most promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has a 7 bp consensus sequence 5-TATA(A/T)A(A/T)-3. TBP binds to TATA box that includes an additional downstream bp.,转录起始点,转

36、录起始位点没有广泛的序列同源性，但第一个碱基为腺嘌呤，而两侧是嘧啶碱基。这个区域被称为起始子（initiator， Inr），序列可表示为PyPyANT/APyPy。Inr元件位于-2+4。仅由Inr元件组成的启动子是具有可被RNA聚合酶II识别的最简单启动子形式 转录起始位点与TATA box一起组成核心启动子； 上游调控元件具有促进转录作用,转录起始点与核心启动子 ( class II recognized by RNA polymerase II ),l Cap site ; initiation point (+1) Capping m7GpppA/G-70 -AUG-(in mRNA

37、),TATA box acts in a similar way to an E. coli promoter 10 sequence to position the RNA Pol II for correct transcription initiation. The spacing but not the sequence between the TATA box and the start site is important. Transcription starts with an adenine 50% of the time.,Some eukaryotic genes cont

38、ain an initiator element instead of a TATA box. The initiator element is located around the transcription start site. Other genes have neither a TATA box nor an initiator element, and usually are transcribed at very low rates.,5.8-3 Upstream regulatory elements,The basal elements (the TATA box and i

39、nitiator elements) : primarily determine the location of the startpoint, and sponsor initiation only at a rather low level. Upstream regulatory elements (URE) such as the SP1 box and CCAAT boxes, greatly increase the frequency of initiation. URE is located within 100-200 bp from the promoter, and pl

40、ays an important role in ensuring efficient transcription.,5.8-4 Enhancers,Sequence elements which can activate transcription from thousands of base pairs upstream or downstream.,General characteristics of Enhancers Exert strong activation of transcription of a linked gene from the correct start site. activate transcription when placed in either orientation with respect to linked genes Able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site. Exert preferential stimulation