分子生物学Chapter 8 Eukaryotic RNA Transcription.docx

1、分子生物学Chapter 8 Eukaryotic RNA TranscriptionChapter 8 Eukaryotic RNA Transcription IntroductionFigure 8-1 Diagram of Eukaryotic Gene1.Basic Features of Eukaryotic transcriton1.1 Three different classes of transcriptionThree different RNA polymerases are responsible for gene transcription:RNA polymera

2、se I (pol I): ribosomal RNAs (5.8S, 18S, 28S rRNA)RNA polymerase II (pol II): mRNAs some small nuclear RNAs (snRNAs)RNA polymerase III (pol III): tRNAs 5S RNA some snRNAs small cytoplasmic RNAs (scRNAs)Basic properties of each polymerase:Enzyme location relative activity a-amanitin sensitivity Pol I

3、 nucleolus 50-70% not inhibitedPol II nucleoplasm 20-40% inhibitedPol III nucleoplasm 10% species-specificRNA polymerase subunitsEukaryotic polymerases has 12 or more different subunit. All contain subunits homologous to the subunits of the E. coli RNA Pol core (2). at least five other subunits are

4、common to three different RNA Pols. Each RNA Pol contain additional four or seven specific subunit.All three polymerase classes. weigh 500,000 D contain 12-16 subunits - some conserved across evolution b - like (200,000 D) b - like (140,000 D) a - like (40,000 D) - some shared among all 3 polymerase

5、s - some unique Figure 10-26. Schematic representation of the subunit structure of yeast nuclear RNA polymerases and comparison with E. coli RNA core polymerase. All three yeast polymerases have four core subunits that exhibit some homology with the , , and subunits in E. coli RNA polymerase. The la

6、rgest subunit (L) of RNA polymerase II also contains an essential C-terminal domain (CTD). RNA polymerases I and III contain the same two nonidentical -like subunits, whereas polymerase II has two copies of a different -like subunit. All three polymerases share five other common subunits (two copies

7、 of the largest of these). In addition, each yeast polymerase contains four to seven unique smaller subunits.1.2 Transcription FactorsThe initial finding that purified eucaryotic RNA polymerase enzymes could not initiate transcription in vitro led to the discovery and purification of additional prot

8、eins, the general transcription factors, required for this process. These proteins were not simply missing subunits of the polymerase; they had to assemble into a complex on the DNA at the promoter in order to recruit the RNA polymerase to this sitetranscription factors: proteins required for bringi

9、ng polymerase to transcription start site to form a preinitiation complex, may not migrate with the polymerase down the DNA template, are not associated with inactive polymerase which are of two types:A) Upstream factors: bind upstream sites to increase efficiency of initiation . regulation of WHICH

10、 promoters are used, but no Temporal or Tissue specificityThe RNA polymerase resulting from the CoreEnzyme plus the TF Upstream Transcription Factors is usually called the Basal Apparatus . or HoloEnzyme, for comparison to prokaryotic RNA polymerases .B )Inducible factors: bind upstream and downstre

11、am sites; Time & Tissue specificityThese Inducible Factors have no cognates in prokaryotic RNA polymerases1.3 DNA Gene Expression (Transcription) SitesPromoter Upstream regulatory element1.4 The process of transcription Transcription by each polymerase involves: 1) recognition of promoter sequences

12、and assembly of an initiation complex at the transcription start site 2) promoter melting 3) initiation and promoter escape 4) elongation (RNA synthesis) 5) termination 2. Pol I transcription ( Reading: Weaver chapter 10 pp. 286-288; chapter 11 pp. 325-331; chapter 16 pp. 498-501Overview: Pol I is r

13、esponsible for transcription of major ribosomal RNAs (note: 5S RNA is transcribed by pol III): 18S RNA 28S RNA 5.8S RNAv accounts for 50-70% of total RNA synthesis - v produces a single transcript that is processed to generate all three RNAsv rRNA gene is multicopy (hundreds-thousands of copies)2.1

14、Gene structurehundreds-thousands of copies of the identical transcription unit, containing 18S - 5.8S - 28S RNA.Ribosomal RNAs are well conserved in eukaryotes, spacers (including length) are not.Nucleolar endonucleases progressively cleave the primary transcript to yield the mature RNAsProduct is a

15、 45S RNA that is processed to give the 18S, 5.8S, and 28S mature rRNAsFig 8-5U3 RNA and associated proteins (U3 snRNP) bind near the 5-end of the primary transcript and direct processing.2.2 Pol I purified from nucleoli (pol II and pol III excluded) unlike pol II (and sometimes pol III), pol I is no

16、t inhibited by a-amanitin high elongation rate (30 nucleotides / second) 500-600 KD, 9-14 subunits2.3 Promoter structurecomparing rRNA gene promoters from different species shows a similar organization but poor conservationconsider mammalian genes map elements by scanning linker insertionsExperiment

17、s identify two regions that are required for full transcription, correspond to binding sites for two different sets of factorsCore promoter: -45 to +20 (+1 is Start Nuc) . G,C-rich 85% seq Upstream Control Element (UCE): -180 to -107 . G,C-rich2.4 Transcription factorsTwo promoter binding activities

18、:UBF: upstream binding factorSL-1: promoter selectivity factor(Sigma-Like factor)All proteins that make up UBF and SL-1 have been recently cloned, expressed recombinantly, shown to be sufficient for pol I transcription.UBF + SL1 + pol I - minimum for transcriptionWhat are UBF and SL-1?UBF: a single

19、94-97 KD protein cloned binds UCE and core promoter DNA binding domain characterized resembles HMG1 (chromain protein) binds in minor groove recognizes structure, not sequence bends linear DNA, induces positive supercoilingSL-1: purified as a macromolecular complex, 200 KD contains 4 proteins: TBP,

20、TAFI110, TAFI63, TAFI48TAFs (TBP Activation Factors); species & promoter specificityTBP TBP is present in both TFIID and SL-12.5 DNA recognitionIn pol II transcription, TBP binds DNA (TATA-box)In pol I transcription, TBP binds TAFIs, TAFIs bind DNA no TATA-box in core promoter both TAF48 and TAF63 c

21、an be cross-linked with DNA TAFI63 contains two Cys2-His2 zinc fingers, mediates interaction with core promoterProcess of factor assembly at the promoter 2.7 Transcription terminationWell understood. In contrast to pol II: termination occurs at well-defined sites polyadenylation does not happenTrans

22、cription terminates in the intergenic spacer (IGS). Specific termination prevents one extending polymerase from occluding downstream initiation complexesTermination studied in mammalian, frog, and yeast systems. as with initiation, mechanism is conserved although factors are species specific termina

23、tion happens at one of eight tandemly repeated termination sites 565 bp downstream of the mature 3-end termination sites, called Sal boxes, share an 18 bp consensus sequence: AGGTCGACCAGATNTCCG Sal I site a single Sal box is sufficient for termination, serves as the binding site for TTFI(transcripti

24、on termination factor I). termination requires TTFI bind the Sal box and that the Sal box be in the correct orientationTTFI causes the polymerase to terminate 10 bp upstream of the Sal box. The in vivo mature end is 21 bp upstream of the Sal box - a pol Iassociated exonuclease cuts 11 bp from the 3-

25、end3. Pol III Transcription3.1 Genes transcribed by RNA polymerase IIISmall RNAs involved in protein synthesis5.8S RNA (ribosomal component)tRNAsSmall RNA involved in mRNA processingU6 snRNA (spliceosome)Small RNA required for protein transport7SL RNA (signal recognition particle)RNA involved in DNA

26、 replicationTelomerase RNARNAs involved in viral gene expressionVA RNAs (Adenovirus)EBER RNAs (Epstein-Barr virus)Unknown function7SK RNAAluIB2 (Rodent)SINE 3.2 Types of pol III promotersHow do you map out promoter structure?Assay for activity - cell-free extracts - Xenopus oocyte injection Linker i

27、nsertion analysis Mutational analysis Deletion analysis5.8S rRNA geneno upstream sequences required for 5S rRNA transcription!Three different types of promoters direct pol III transcriptionType 1, Type 2 genes: intragenic promoters, internal control regions (ICRs) are located downstream of transcrip

28、tion startType 3 genes: extragenic promoters, upstream control regions including TATA boxesNote: the type distinctions are not absolute, for instance -upstream sequences in type 1 and 2 genes can alter transcription levels -X. laevis tRNA(Ser)Sec has both intra- and extragenic promoter elements3.2.1

29、 Type 1: e.g. Xenopus laevis 5S RNA -3 types of 5S genes: adult 400 genes major oocyte 20,000 genes trace oocyte 1,000 genes -ICR spans 45 bp, runs from +50 to +90 -3 distinct internal promoter elements A-box C-box intermediate element (IE) 3.2.2 Type 2: represented by tRNA genes -ICR (+10/+65) -2 d

30、istinct promoter elements A-box - corresponds to D-loop sequence B-box - corresponds to T-loop sequence In type 1 genes: A-box is at +50 In type 2 genes: A-box is at +10, determines transcription start site Spacing between A and B varies (30-60 nts.) 5S A-box and tRNA A-box are interchangeable3.2.3

31、Type 3 genes: e.g. U6 snRNA, 7SK RNA -no ICR -multiple upstream promoter elements -TATA box (-30) -proximal sequence element (-60) -distal sequence element (-200) DSE is made up by two sequence motifs -GC box: GGGCGG -octamer motif: ATGCAAAT OCT binds Octamer Binding Factor (OTF-1)Stimulatory sites for RNA PolIII Similar to structure for other U RNAs that are transcr