七年制分子生物復(fù)習(xí)

1、Replication復(fù)制, transcription轉(zhuǎn)錄 and translation轉(zhuǎn)移Questions:1. What is semi-discontinuous replication?The leading strand copies continuouslyThe lagging strand copies in segments (Okazaki fragments) which must be joined2. Which enzymes and protein participate in DNA synthesis? What functions do they ex

2、ert in the replication process?原核Initiation of Replication：1）DnaA is a replication initiation factor which promotes the unwinding of DNA at oriC during DNA replication in E.coli.2）DnaB (helicase) moves along a DNA strand opening up the duplex to melt and separate the DNA strands.bind to DnaG initiat

3、ing primer synthesis.3）Single-stranded binding proteins (SSBs) stabilize single-stranded DNA，prevent from reannealing(不覆蓋堿基，不影響ssDNA作為復(fù)制模板)4）DnaG(Primase) catalyzes synthesis of short RNA molecules that function as primers for DNA synthesis in E. coli. Primase binds to DnaB protein at oriC and forms

4、 a primosome.（可在隨從鏈中移動）5）Type I topoisomerase cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is religeted. Type II topoisomerase cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then religetes the cut strands.Topoisomeras

5、e removes supercoils produced by DNA unwinding at the replication forkElongation of Replication：1） DNA polymerase:The enzymes that replicate DNA .The incoming base is selected within the DNA polymerases active site.Chain growth is in the 5 to 3 direction.Pol I: Gap filling during DNA synthesis and r

6、epair, removal of RNA primers Pol II: Involved in DNA synthesis of damaged templates Pol III: Functional polymerase at the replication fork Termination of Replication:1） RNAse: remove RNA primers 2） DNA polymerase I: The RNA primer is removed(the last nuclotide) and replaced with DNA by polymerase I

7、3） DNA ligase: seals the gaps between Okazaki fragments with a phosphodiester bond真核：1）DNA polymerases in eukaryotic cellshas a primase subunit and functions in initiations synthesizes the RNA primers and a stretch of DNA sequences, has no proofreading activity.the principal DNA polymerase in eukary

8、otic cells has proofreading activity.its precise function is unclear; sensor for DNA damage checkpoint controlDNA repairmitochondrial DNA replication2） RPA(replication protein A) binds to the melted single-stranded DNA.3）RFC(replication factor C) RFC is the eukaryotic counterpart of the prokaryotic

9、-complex. RFC can stimulate Pol a3） PCNA (proliferating cell nuclear antigen, similar to eukaryotic sliding DNA clamp) displaces Pol a-primase.It can clamp DNA polymerase to the DNA.4） T antigen：It is a multifunctional site-specific DNA binding protein encoded by SV40 DNA, binds to the origin (as Dn

10、aA) and melts the duplex DNA (as DnaB)Prokaryotic vs. Eukaryotic cells Replication Enzymes：Prokaryotic eukaryoticDnaB Helicase Helicase T-Ag (SV40)SSB RPA- Complex Clamp Loader RFC Sliding Clamp PCNAPrimase (DnaG) Primase activity of Pol a Pol III Pol , then Pol d3. Please describe the process of DN

11、A synthesis.原核in E.coli:1)DnaA protein binds to the 9-mer region in oriC and forming a multimeric complex with 10-20 protein subunits. Binding requires ATP.2)Further addition of ATP was observed to result in a melting and opening up of the DNA duplex in the oriC region.3) After helicase/DnaC binds t

12、o the DNA, the dnaC protein is released.4) Single-stranded DNA-binding (SSB) proteins stabilize the single-stranded template DNA during the process. 5)DNA primase next binds to helicase producing a complex called a primosome Primase synthesizes a short RNA primer, to which DNA polymerase III adds nu

13、cleotides.6)Polymerase III adds nucleotides 5 to 3 on both strands beginning at the RNA primer.-protein:使helicase夾緊 complex (clamp loader):load sliding clamp on DNA strandSliding clamp:固定識別為點，使DNA pol 與DNA 有高親和力7)The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed

14、with DNA ligase. 真核：Model of in vitro replication of SV40 DNA by eukaryotic enzymes:1）T antigen (Tag) binds and unwinds replication origin;2）RPA binds to single-stranded DNA;3）Pol -primase synthesizes the primers (RNA + DNA).4.）RFC loads PCNA to the template.5.）PCNA displaces Pol -primase and functi

15、ons as a DNA clamp;6） Pol replaces Pol -primase and further extends the DNA strands.4. What is the meaning of wobble base pairing and answer the manner of base pairing?5. Try to compare the differences and similarities between the transcription and translation.Similarity between replication and tran

16、scription:1)Both processes use DNA as the template.2)Phosphodiester bonds are formed in both cases. 3)Both synthesis directions are from 5 to 3. Differences between replication and transcription:6. Describe E.coli RNA polymerase.1)RNA polymerases performs essentially the same reaction in all cells.

17、2)Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III3) Holoenzyme（全酶）= factor + core enzyme The core enzyme alone synthesizes RNA factor: Recognise promoter, initiates transcription only at promoters. 進(jìn)入延長階段后與core enzyme 解離（全酶的不同是因為亞基的不同） cor

18、e enzyme: : fix DNA strand together with pol : decide which gene is to be transcripted: unclear7. Please describe the process of transcription in prokaryotes.1)Transcription initiation：在起始點附近解旋，從起始點開始轉(zhuǎn)錄Transcription bubble: 17-19bp2)Transcription elongation: polymerase advances 35down template stran

19、d,melting duplex DNA and adding rNTPs to growing RNA.3)Transcription termination: at transcription stop site,polymerase releases completed RNA and dissociates from DNA.4)Transcription is terminated by signals within the RNA sequence:具體見PPT8. Please describe the processing and modification of mRNA pr

20、ecursors in eukaryotes1)RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genesRNA Pol I transcribe the large ribosomal RNA precursor geneRNA Pol III transcribe

21、tRNA gene, some small nuclear RNA genes and 5S rRNA genes）2)-Amanitin is an inhibitor of RNA polymerase II. This mechanism makes it a deadly toxin.-Amanitin can also be used to determine which types of RNA polymerase are present. This is done by testing the sensitivity of the polymerase in the prese

22、nce of -amanitin. 3) The carboxy-terminal domain (CTD) of RNA polymerase II typically consists of repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (pol 1,3 沒有)（不同生物共有序列的重復(fù)程度不同）1. 4) RNA Pol II forms a pre-initiation complex with GTFs at the promoter：The involved GTFIIs (general transcription fact

23、or for Pol II)TFIID=TBP (TATA box binding protein) + TAFs (TBP association factors) TFIIA, B, F, E, H5）process：TBP in TFIID binds to the TATA box(TBP: TATA box bing protein)TFIIA and TFIIB are recruited forming a TBP-TFIIB-promoter complex RNA Pol II-TFIIF complex is then recruitedTFIIE and TFIIH th

24、en bind to Pol II to form the pre-initiation complex (轉(zhuǎn)錄前復(fù)合物)Promoter melting using energy from ATP hydrolysis by TFIIH Promoter escapes after the phosphorylation of the CTD tail（repeat sequence）Elongation:must deal with histons in its path(隨著RNA pol的移動，helix向前走，但不會解開，繞過核小體nucleosome)Transcription termination: The termination sequence is AATAAA followed by GT repeats. The termination is closely related to the post-transcriptional modification.(轉(zhuǎn)錄終止后，RNA pol去磷酸化)5） The functio