1. Transcription(RNAbiosynthesis)

2. TranscriptionThe synthesis of RNA m olecules using DNAstrands as the templates so that the geneticinformation can be transferred from DNA toRNA. 3. OverviewThere are four major types of RNA molecules:a. Messenger RNA (mRNA) encodes the aminoacid sequence of a polypeptide.b. Transfer RNA (tRNA) brings amino acids toribosomes during translation.c. Ribosomal RNA (rRNA) combines withproteins to form a ribosome, the catalyst fortranslation.d. Small nuclear RNA (snRNA) combines withproteins to form complexes used in eukaryoticRNA processing. 4. The Transcription Process RNASynthesis1. Transcription, or gene expression, is regulated by generegulatory elements associated with each gene.2. DNA unwinds in the region next to the gene.3. RNA is transcribed 5-to-3. The template DNA strand isread 3-to-5. Its complementary DNA, the nontemplatestrand, has the same polarity as the RNA.4. RNA polymerization is similar to DNA synthesis, except:a. The precursors are NTPs (not dNTPs).b. No primer is needed to initiate synthesis.d. Uracil is inserted instead of thymine. 5. Similarity betweenreplication and transcription Both processes use DNA as the template. Phosphodiester bonds are formed in bothcases. Both synthesis directions are from 5 to 3. 6. The RNA polymerase-catalyzed synthesis ofRNA on a DNA template strand 7. Differences betweenreplication and transcriptionreplication transcriptiontemplate double strands single strandsubstrate dNTP NTPprimer yes noEnzyme DNA polymerase RNA polymeraseproduct dsDNA ssRNAbase pair A-T, G-C A-U, T-A, G-C 8. The Transcription ProcessInitiation of Transcription at PromotersTranscription is divided into three steps for bothprokaryotes and eukaryotes.initiation, elongation and termination.The process of elongation is highly conservedbetween prokaryotes and eukaryotes, butinitiation and termination are somewhatdifferent. 9. Template and Enzymes 10. The whole genome of DNA needs to bereplicated, but only small portion of genomeis transcribed in response to thedevelopment requirement, physiological needand environmental changes. DNA regions that can be transcribed intoRNA are called structural genes. 11. TemplateThe template strand is the strand from which the RNA isactually transcribed. It is also termed as antisense strand.The coding strand is the strand whose base sequencespecifies the amino acid sequence of the encoded protein.Therefore, it is also called as sense strand.codingstrandG C 5' A G T A C A T G T C 3'3' C G T C A T G T A C A G 5' templatestrandtranscription5' G C A G U A C A U G U C 3' RNA 12. Asymmetric transcription Only the template strand is used for the transcription, butthe coding strand is not. The transcription direction on different strands isopposite. This feature is referred to as the asymmetrictranscription.5'3'3'5' 13. Organization of coding information inthe adenovirus genome 14. RNA Polymerase The enzyme responsible for the RNAsynthesis is DNA-dependent RNApolymerase. The prokaryotic RNA polymerase is amultiple-subunit protein of ~480kD. Eukaryotic systems have three kinds ofRNA polymerases, each of which is amultiple-subunit protein and responsible fortranscription of different RNAs. 15. RNA-pol of E. ColiThe holoenzyme of RNA-p ol in E.coli consists of 5different subunits: a2 b b ws.core enzymeb ba awssubunit MW functiona 36512 Determine the DNA to betranscribedb 150618 Catalyze polymerizationb 155613 Bind & open DNA templates 70263 Recognize the promoterfor synthesis initiation 16. Rifampicin, a therapeutic drug fortuberculosis treatment, can bindspecifically to the b subunit of RNA-pol,and inhibit the RNA synthesis. RNA-pol of other prokaryotic systems issimilar to that of E. coli in structure andfunctions. 17. RNA-pol of eukaryotesRNA-pol I II IIIproducts 45S rRNA hnRNA5S rRNAtRNAsnRNASensitivityto Amanitin No high moderateAmanitin is a specific inhibitor of RNA-pol.from a mushroom, inhibits Pol II, and PolIII at higher concentrations. 18. Recognition of Origins Each transcriptable region is calledoperon. One operon includes several structuralgenes and upstream regulatory sequences(or regulatory regions). The promoter is the DNA sequence thatRNA-pol can bind. It is the key point forthe transcription control. 19. 5'3'3'5'Promoterregulatorysequences structural genepRroNmA-opotol r 20. 5'3'3'5'Prokaryotic promoter-50 -40 -30 -20 -10 1 10-10 startregionT A T A A TA T A T T A(Pribnow box)-35regionT T G A C AA A C T G TConsensus sequence 21. Consensus Sequence 22. The -35 region of TTGACA sequence is therecognition site and the binding site of RNA-pol. The -10 region of TATAAT is the region atwhich a stable complex of DNA and RNA-polis formed. 23. Transcription Process 24. General concepts Three phases: initiation, elongation,and termination. The prokaryotic RNA-pol can bind tothe DNA template directly in thetranscription process. The eukaryotic RNA-pol requires co-factorsto bind to the DNA templatetogether in the transcription process. 25. Transcription of Prokaryotes Initiation phase: RNA-pol recognizesthe promoter and starts thetranscription. Elongation phase: the RNA strand iscontinuously growing. Termination phase: the RNA-pol stopssynthesis and the nascent RNA isseparated from the DNA template. 26. a. Initiation RNA-pol recognizes the TTGACAregion, and slides to the TATAATregion, then opens the DNA duplex. The unwound region is about 171bp. 27. The first nucleotide on RNA transcriptis always purine triphosphate. GTP ismore often than ATP. The pppGpN-OH structure remains onthe RNA transcript until the RNAsynthesis is completed. The three molecules form atranscription initiation complex.RNA-pol (a2bbs) - DNA - pppGpN- OH 3 28. No primer is needed for RNAsynthesis. The s subunit falls off from the RNA-polonce the first 3,5 phosphodiesterbond is formed. The core enzyme moves along theDNA template to enter the elongationphase. 29. b. Elongation The release of the s subunit causesthe conformational change of thecore enzyme. The core enzymeslides on the DNA template towardthe 3 end. Free NTPs are added sequentially tothe 3 -OH of the nascent RNA strand. 30. RNA-pol, DNA seg ment of ~40nt andthe nascent RNA form a complexcalled the transcription bubble. The 3 segment of the nascent RNAhybridizes with the DNA template,and its 5 end extends out thetranscription bubble as the synthesisis processing. 31. Transcription bubble 32. RNA-pol of E. Coli 33. RNA-pol of E. Coli 34. Simultaneoustranscriptions andtranslation 35. c. Termination The RNA-pol stops moving on theDNA template. The RNA transcriptfalls off from the transcriptioncomplex. The termination occurs in either r-dependent or r -independentmanner. 36. The termination function of r factorThe r factor, a hexamer, is a ATPaseand a helicase. 37. r-independent termination The termination signal is a stretch of30-40 nucleotides on the RNAtranscript, consisting of many GCfollowed by a series of U. The sequence specificity of thisnascent RNA transcript will formparticular stem-loop structures toterminate the transcription. 38. DNA5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 35TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3RNAUUUU...UUUU... 39. Stem-loop disruption The stem-loop structure alters theconformation of RNA-pol, leading tothe pause of the RNA-pol moving. Then the competition of the RNA-RNAhybrid and the DNA-DNA hybridreduces the DNA-RNA hybridstability, and causes thetranscription complex dissociated. Among all the base pairings, themost unstable one is rU:dA. 40. Transcription of Eukaryotesa. Initiation Transcription initiation needspromoter and upstream regulatoryregions. The cis-acting elements are thespecific sequences on the DNAtemplate that regulate thetranscription of one or more genes. 41. Cis-acting elementstructural genecis-acting elementGCGC CAAT TATAexon intron exonstartCAAT boxenhancerGCboxTATA box (Hogness box) 42. TATA box 43. Transcription factors RNA-pol does not bind the promoterdirectly. RNA-pol II associates with sixtranscription factors, TFII A - TFII H. The trans-acting factors are theproteins that recognize and binddirectly or indirectly cis-actingelements and regulate its activity. 44. TF for eukaryotic transcription 45. Pre-initiation complex (PIC) TBP of TFII D binds TATA TFII A and TFII B bind TFII D TFII F-RNA-pol complex binds TFII B TFII F and TFII E open the dsDNA (helicase and ATPase) TFII H: completion of PICRNA pol IITF II FTBP TAFTATADNATF IIATF IIBTF II ETF II H 46. Transcription factors and the pre-initiation complex 47. Phosphorylation of RNA-pol TF II H is of protein kinase activity tophosphorylate CTD of RNA-pol. (CTDis the C-terminal domain of RNA-pol) Only the RNA-pol can move towardthe downstream, starting theelongation phase. Most of the TFs fall off from PICduring the elongation phase. 48. b. Elongation The elongation is similar to that ofprokaryotes. The transcription and translation donot take place simultaneously sincethey are separated by nuclearmembrane. 49. RNA-PolnucleosomeRNA-PolRNA-Polmovingdirection 50. c. Termination When the RNA Polymerase transcribes theterminator region of the DNA, the polymerasereleases the mRNA The termination sequence is AATAAAfollowed by GT repeats. In eukaryotes, the release factor (eRF) whichrecognizes all three stop codons. The overallprocess of termination is similar inprokaryotes, but 3 release factors exist, RF1,RF and RF3. 51. Post-TranscriptionalModification 52. The nascent RNA, also known asprimary transcript, needs to bemodified to become functionaltRNAs, rRNAs, and mRNAs. The modification is critical toeukaryotic systems. 53. Modification of hnRNA Primary transcripts of mRNA are called asheteronuclear RNA (hnRNA). hnRNA are larger than matured mRNA by manyfolds. Modification includes Capping at the 5- end Tailing at the 3- end mRNA splicing RNA editing 54. a. Capping at the 5- endOH OHOH2N NO PCH3OCH2ON NHNO OHO POONNH2AAAAA-OHOPi5'3'ON H2CHN NOOOO POOO PO5'm7GpppGp---- 55. The 5- cap structure is found on hnRNAtoo. The capping process occurs innuclei. The cap structure of mRNA will berecognized by the cap-binding proteinrequired for translation. The capping occurs prior to the splicing.addition of 5cap:Prevents unravelingHelps ribosome attach 56. b. Poly-A tailing at 3 - end There is no poly(dT) sequenceon the DNA template. Thetailing process dose not dependon the template. The tailing process occurs priorto the splicing. The tailing process takes placein the nuclei.addition of poly A tailPrevents unravelingAssists in the export of mRNA fromnucleus 57. c. mRNA splicingDNAmRNAThe matured mRNAs are much shorter thanthe DNA templates. 58. Split geneThe structural genes are composed ofcoding and non-coding regions thatare alternatively separated.7 700 bpL 1 2 3 4 5 6 7A B C D E F GA~G no-coding region 1~7 coding region 59. Exon and intronExons are the coding sequences thatappear on split genes and primarytranscripts, and will be expressed tomatured mRNA.Introns are the non-coding sequencesthat are transcripted into primarymRNAs, and will be cleaved out in thelater splicing process. 60. mRNA splicing 61. Splicing mechanism 62. lariat 63. TGGCNNAGTGC GGTTCGANNCCRNA-pol IIItRNA precursorDNAPrecursor transcription 64. Twice transesterificationintron5'exon 3'exon5' U pA G pU 3'pG-OHpGpAfirst transesterification5' UOH G pU 3'second transesterificationpGpA5' U pU 3'GOH5'3' 65. d. mRNA editing Taking place at the transcriptionlevel One gene responsible for more thanone proteins Significance: gene sequences, afterpost-transcriptional modification,can be multiple purposedifferentiation. 66. Different pathway of apo BHuman apo BgenehnRNA (14 500 base)CAA to UAAliverapo B100500 kD intestineapo B48240 kD 67. Modification of tRNA 68. CleavageRNAase Pendonucleaseligase 69. Addition of CCA-OHtRNA nucleotidyltransferaseATP ADP 70. Base modification113241. MethylationAmA, GmG2. ReductionUDHU3. TransversionU4. DeaminationAILittle is known about the role of specific base modifications of transfer RNAs 71. Modification of rRNA 45S transcript in nucleus is the precursor of 3 kindsof rRNAs. The matured rRNA will be assembled with ribosomalproteins to form ribosomes that are exported tocytosolic space.rRNA18S 5.8S 28Stranscriptionsplicing45S-rRNA18S-rRNA5.8S and 28S-rRNA 72. Prokaryotic Regulation Control of transcription initiation can be: positive control increases transcription whenactivators bind DNA negative control reduces transcription whenrepressors bind to DNA regulatory regionscalled operators 73. Prokaryotic Regulation Prokaryotic cells often respond to their environment bychanges in gene expression. Genes involved in the same metabolic pathway areorganized in operons. A regulatory sequence adjacent to such a unit determineswhether it is transcribed - this is the operator Some operons are induced when the metabolic pathway isneeded. Some operons are repressed when the metabolic pathwayis no longer needed. 74. Prokaryotic Regulation The lac operon contains genes for the use oflactose as an energy source. Regulatory regions of the operon include the CAP(catabolite activator protein) binding site,promoter, and the operator. The coding region contains genes for 3 enzymes: b-galactosidase, permease, and transacetylase 75. Prokaryotic Regulation The lac operon is negatively regulated by arepressor protein: lac repressor binds to the operator to blocktranscription in the presence of lactose, an inducer moleculebinds to the repressor protein repressor can no longer bind to operator transcription proceeds 76. Prokaryotic Regulation In the presence of both glucose and lactose,bacterial cells prefer to use glucose. Glucose prevents induction of the lac operon. binding of CAP cAMP complex to the CAPbinding site is required for induction of the lac operon high glucose levels cause low cAMP levels high glucose low cAMP no induction 77. Prokaryotic Regulation The trp operon encodes genes for thebiosynthesis of tryptophan. The operon is not expressed when the cellcontains sufficient amounts of tryptophan. The operon is expressed when levels oftryptophan are low. 78. Prokaryotic Regulation The trp operon is negatively regulated bythe trp repressor protein trp repressor binds to the operator to blocktranscription binding of repressor to the operator requires acorepressor which is tryptophan low levels of tryptophan prevent the repressorfrom binding to the operator 79. Eukaryotic Regulation Controlling the expression of eukaryoticgenes requires transcription factors. general transcription factors are required fortranscription initiation required for proper binding of RNA polymerase tothe DNA specific transcription factors increasetranscription in certain cells or in response tosignals 80. Eukaryotic Transcription General transcription factors bind to the promoter region ofthe gene. RNA polymerase II then binds to the promoter to begintranscription at the start site (+1). Enhancers are DNA sequences to which specifictranscription factors (activators) bind to increase the rate oftranscription. 81. Mechanisms of enhancer action DNA looping model postulates that proteins bound to a distantenhancer interact directly with components of the transcriptioninitiation complex, by looping out the DNA An enhancer noncovalently linked to a promoter via a protein bridgeis functional Enhancer function requires close proximity to the promoter Enhancers do not serve as entry sites for RNA polymerase II 82. Eukaryotic Transcription Coactivators and mediators are alsorequired for the function of transcriptionfactors. coactivators and mediators bind to transcriptionfactors and bind to other parts of thetranscription apparatus 83. Posttranscriptional Regulation Control of gene expression usually involves thecontrol of transcription initiation. But gene expression can be controlled aftertranscription, with mechanisms such as: RNA interference alternative splicing RNA editing mRNA degradation 84. Posttranscriptional Regulation RNA interference involves the use of smallRNA molecules The enzyme Dicer chops double strandedRNA into small pieces of RNA micro-RNAs bind to complementary RNA toprevent translation small interfering RNAs degrade particularmRNAs before translation 85. Micro RNA (miRNA) Production of a functional miRNA begins in thenucleus Ends in the cytoplasm with a ~22 nt RNA thatfunctions to repress gene expression miRNA loaded into RNA induced silencingcomplex (RISC) RISC is targeted to repress the expression of genesbased on sequence complementarity to the miRNA 86. CytoplasmDicerPri-microRNAMature miRNARISC mRNARNA Polymerase IImicroRNA geneRISCmRNA cleavagemRNARISC RISCInhibition of translationNucleusDroshaExportin 5Pre-microRNA 87. siRNA RNA interference involves the productionof siRNAs Production similar to miRNAs but siRNAsarise from long double-stranded RNA Dicer cuts yield multiple siRNAs to loadinto RISC (RNA-induced silencingcomplex) Target mRNA is cleaved 88. miRNA or siRNA? Biogenesis of both miRNA and siRNAinvolves cleavage by Dicer andincorporation into a RISC complex Main difference is target miRNA repress genes different from their origin Endogenous siRNAs tend to repress genes that theywere derived from 89. Exogenous dsRNA, transposon, virusPPRepeated cuttingby dicerPPPPPP siRNAsAgo +RISCAgoRISCsiRNAin RISCCleavage of target mRNAmRNA 90. Alternative splicing Introns are spliced out of pre-mRNAs to producethe mature mRNA Tissue-specific alternative splicing The mature mRNAs in each tissue possessdifferent exons, resulting in differentpolypeptide products from the same gene. Determined by tissue-specific factors thatregulate the processing of the primary transcript 91. Why bother with introns? Introns may regulate gene activity and thepassage of mRNA into the cytoplasm Genes may play roles in multiple proteins,introns may enable a gene to be diverse infunction May increase recombination of geneticmaterial (easier to cut and paste) 92. RNA editing Creates mature mRNA that are not trulyencoded by the genome Involves chemical modification of a base tochange its base-pairing properties Apolipoprotein B exists in 2 isoforms One isoform is produced by editing the mRNAto create a stop codon This RNA editing is tissue-specific 93. Initiation of translation can be controlled Ferritin mRNA only translated if iron present Mature mRNA molecules have varioushalf-lives depending on the gene and thelocation (tissue) of expression Target near poly-A tail can cause loss of the tail anddestabilizationThe amount of polypeptide produced from a particulargene can be influenced by the half-life of the mRNAmolecules. 94. DNA2. RNA splicingGene expressioncan be controlledby altering therate of splicing ineukaryotes.Alternative splicingcan producemultiple mRNAsfrom one gene.5 capCutintron3 poly-A tailMature RNA transcipt ExonsIntronsRNA polymerase II35 Primary RNA transcript1. Initiation oftranscriptionMost control ofgene expressionis achievedby regulatingthe frequencyof transcriptioninitiation. 95. 3. Passage throughthe nuclearmembraneGene expressioncan be regulatedby controllingaccess to orefficiency oftransport channels.4. Protein synthesisMany proteins takepart in thetranslation process,and regulationof the availabilityof any of them altersthe rate of geneexpression byspeeding or slowingprotein synthesis.35NuclearporeSmallsubunit5 capmRNALarge3 poly-A tail subunit 96. 5. RNA interferenceGene expressionis regulated bysmall RNAs. Proteincomplexescontaining siRNAand miRNA targetspecific mRNAs fordestruction or inhibittheir translation.Completedpolypeptidechain6. PosttranslationalmodificationPhosphorylationor other chemicalmodifications canalter the activityof a protein afterit is produced.RISCPP 97. Ribozyme The rRNA precursor of tetrahymenahas the activity of self-splicing. The catalytic RNA is called ribozyme. Self-splicing happened often for intron Iand intron II. Both the catalytic domain and thesubstrate locate on the same molecule,and form a hammer-head structure. At least 13 nucleotides are conserved. 98. Hammer-head 99. Significance of ribozyme Be a supplement to the central dogma Redefine the enzymology Provide a new insights for the origin oflife Be useful in designing the artificialribozymes as the therapeutical agents 100. Retrovirus Replication Cycleds DNAhost DNA ProvirusPackaging into virus ; budding 101. Sample questions Which of the following enzyme is used forsynthesis of RNA under the direction ofDNA? A. RNA polymerase B. DNA ligase C. DNA polymerase D. RNA ligase 102. Which of the following is a product oftranscription? A. mRNA B. tRNA C. rRNA D. all of these 103. Recognition/binding site of RNApolymerase is called A. receptor B. promoter C. facilitator D. terminator 104. An mRNA transcript of a gene contains A. a start codon B. a stop codon C. a terminator D. all of these 105. The components found in all prokaryotictranscription terminators is A. a poly-U region B. Rho factor C. a hairpin structure D. none of these 106. Where in the cell is the DNA transcribed intomRNA? A.Cytoplasm B. Nucleus C. Golgi D.Cell cytoskeleton 107. Which of the following does NOT happen during hnRNAprocessing? A. Introns are spliced out. B. A 7-methylguanosine cap is added to the 5' end of the RNA. C. A poly A tail is added. D. Ribosomes bind and begin translation. 108. Since the two strands of the DNA molecule arecomplementary, for any given gene: A. The RNA polymerase can bind to either strand. B. Only one strand actually carries the genetic code for aparticular gene. C. Each gene possesses an exact replica so that nomutation occurs. D. A gene transcribed in the 5 to 3 direction on onestrand can be transcribed in the 3 to 5 direction on theother strand.