2. Molecular Biology Course Section K: Transcription in prokaryotes

3. K1 Basic principles of transcription K2 Escherichia coli RNA polymerase K3 The E. coli  70 promoter K4 transcription process. An overview, the process of RNA synthesis ( initiation, elongation, termination) Properties,  subunit,  subunit,  ’ subunit, sigma (  ) factor Promoter,  70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency Promoter binding, unwinding, RNA chain initiation, elongation, termination (  factor) Section K: Transcription in prokaryotes

4. Molecular Biology Course K1: Basic principles of transcription 1.Transcription: an overview (comparison with replication) 2.The process of RNA synthesis: initiation, elongation, termination

5. Molecular Biology Course K1-1: Transcription: an overview

6. 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, the coding region is downstream of the initiation codon. Gene X Primary transcript m7 Gppp AAAAAn +1 upstream downstream mRNA

7. Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon 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

8. RNA Terminator is a sequence of DNA, represented 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 transcription unit.

9. 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 K1: Basic principles of transcription

10. Replication-synthesis of the leading strand the same direction as the replication fork moves Review of replication

11. Replication- Synthesis of the Okazaki fragments Opposite to the replication fork movement Review of replication

12. Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)

13. Transcription K1: Basic principles of transcription

14. 1.RNA synthesis occurs in the 5’  3’ direction and its sequence corresponds to the sense strand (coding strand). 2.The template of RNA synthesis is the antisense strand (template strand). 3.Phosphodiester bonds: same as in DNA 4.Necessary components: RNA polymerase, transcription factors, rNTPs, promoter & terminator/template K1: Basic principles of transcription

15. K1-2: The process of RNA synthesis 1.initiation 2.elongation 3.termination K1: Basic principles of transcription

16. Flowchart of RNA synthesis Back 1Back 1, 22

17. K1: Basic principles of transcription Promoter Terminator Transcribed region Sense strand Antisense strand DNA RNA Transcription +1 Fig. 2. Structure of a typical transcription unit Is transcribed region equal to coding region? Why?

18. K1: Basic principles of transcription Initiation (template recognition) 1.Binding of an RNA polymerase to the dsDNA 2.Slide to find the promoter 3.Unwind the DNA helix 4.Synthesis of the RNA strand at the start site (initiation site), this position called position +1 Link

19. K1: Basic principles of transcription Elongation Covalently adds ribonucleotides to the 3’-end of the growing RNA chain. The RNA polymerase extend the growing RNA chain in the direction of 5’  3’ The enzyme itself moves in 3’ to 5’ along the antisense DNA strand. Link

20. K1: Basic principles of transcription Termination Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site. Terminator: often contains self- complementary regions which can form a stem-loop or hairpin structure in the RNA products (see K4 for details)

21. Terminator structure

22. Molecular Biology Course K2 Escherichia coli RNA polymerase 1.E. coli RNA polymerase  subunit  subunit  ’ subunit 5.sigma (  ) factor

23. K2: E. coli RNA polymerase K2-1 E. coli RNA polymerase Synthesis of single-stranded RNA from DNA template.

24. 1.Requires no primer for polymerization 2.Requires DNA for activity and is most active with a double-stranded DNA as template. 3.5’  3’ synthesis 4.Require Mg 2+ for RNA synthesis activity 5.lacks 3’  5’ exonuclease activity, and the error rate of nucleotides incorporation is 10 -4 to 10 -5. Is this accuracy good enough for gene expression?? 6. usually are multisubunit enzyme. K2: E. coli RNA polymerase RNA polymerase (NMP)n + NTP  (NMP)n+1 + PPi

25. E. coli polymerase 1.E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. 2.One of the largest enzyme in the cells 3.Consists of at least 5 subunits in the holoenzyme, 2 alpha (  ), and 1 of beta (  ), beta prime (  ’), omega (  ) and sigma (  ) subunits 4.Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp. 5.RNA synthesis rate: 40 nt per second at 37 o C K2: E. coli RNA polymerase

26. E. coli RNA polymerase Both initiation & elongation Initiation only 36.5 KD 151 KD 155 KD 11 KD 70 KD K2: E. coli RNA polymerase

27. The polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesize RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters. RNA polymerase differs from organism to organism K2: E. coli RNA polymerase

28. K2-2:  subunit K2: E. coli RNA polymerase

29. E. coli polymerase:  subunit Two identical subunits in the core enzyme Encoded by the rpoA gene Required for assembly of the core enzyme Plays a role in promoter recognition. Experiment: When phage T4 infects E. coli, the α subunit is modified by ADP-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme. plays a role in the interaction of RNA polymerase with some regulatory factors K2: E. coli RNA polymerase

30. K2-3&4:  and  ’ subunit K2: E. coli RNA polymerase

31.  is encoded by rpoB gene, and  ’ is encoded by rpoC gene. 2.Make up the catalytic center of the RNA polymerase 3.Their sequences are related to those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features to the actions of all RNA polymerases. 4.The  subunit can be crosslinked to the template DNA, the product RNA, and the substrate ribonucleotides; mutations in rpoB affect all stages of transcription. Mutations in rpoC show that  ’ also is involved at all stages.

32.  subunit may contain two domains responsible for transcription initiation and elongation Rifampicin ( 利福平 ) ： has been shown to bind to the β subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation 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. K2: E. coli RNA polymerase

33.  ’ subunit Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase Hyparin ( 肝素 ) ： binds to the  ’ subunit and inhibits transcription in vitro. Hyparin competes with DNA for binding to the polymerase. 2.  ’ subunit may be responsible for binding to the template DNA. K2: E. coli RNA polymerase

34. K2-5: Sigma (  ) factor K2: E. coli RNA polymerase

35. 1.Many prokaryotes contain multiple  factors to recognize different promoters. The most common  factor in E. coli is  70. 2.Binding of the  factor converts the core RNA pol into the holoenzyme.  factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (10 4 ) and increasing the affinity for the corresponding promoter  factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) 5.Less amount of  factor is required in cells than that of the other subunits of the RNA pol.

36. Molecular Biology Course K3: The E. coli  70 promoter 1. Promoter  70 size 3. -10 sequence 4. -35 sequence 5. transcription start site 6. promoter efficiency

37. K3: The E. coli  70 promoter K3-1: Promoter 1.The specific short conserved DNA sequences: 2.upstream from the transcribed sequence, and thus assigned a negative number (location) 3.required for specific binding of RNA Pol. and transcription initiation (function) 4.Were first characterized through mutations that enhance or diminish the rate of transcription of gene

38. Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription. Promoter Terminator Transcribed region Sense strand Antisense strand DNA RNA Transcription +1 K3: The E. coli  70 promoter

40. K3-2,3&4:  70 promoter K3: The E. coli  70 promoter

41. Consists of a sequence of between 40 and 60 bp -55 to +20: bound by the polymerase -20 to +20: tightly associated with the polymerase and protected from nuclease digestion by DNaseΙ(see the supplemental) Up to position –40: critical for promoter function (mutagenesis analysis) -10 and –35 sequence: 6 bp each, particularly important for promoter function in E. coli ---5-8 bp--- G C T A TTGACA TATAAT -----16-18 bp------- +1 -35 sequence -10 sequence K3: The E. coli  70 promoter

42. -10 sequence (Pribonow box) 1.The most conserved sequence in  70 promoters at which DNA unwinding is initiated by RNA Pol. 2.A 6 bp sequence which is centered at around the –10 position, and is found in the promoters of many different E. coli gene. 3.The consensus sequence is TATAAT. The first two bases (TA) and the final T are most highly conserved. 4.This hexamer is separated by between 5 and 8 bp from position +1, and the distance is critical. K3: The E. coli  70 promoter

43. -35 sequence: enhances recognition and interaction with the polymerase  factor A conserved hexamer sequence around position –35 A consensus sequence of TTGACA The first three positions (TTG) are the most conserved among E. coli promoters. Separated by 16-18 bp from the –10 box in 90% of all promoters K3: The E. coli  70 promoter

44. 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 material

45. Supplemental material Footprinting

46. Supplemental material Footprinting

47. K3-5: Transcription start site K3: The E. coli  70 promoter 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)

48. The sequences of five E. coli promoters K3: The E. coli  70 promoter

49. K3-6: promoter efficiency K3: The E. coli  70 promoter There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold.

50. 1.The –35 sequence constitutes a recognition region which enhances recognition and interaction with the polymerase  factor. 2.The -10 sequence is important for DNA unwinding. 3.The sequence around the start site influence initiation efficiency. 4.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.

51. Some promoter sequence are not sufficiently 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 order to enhance polymerase binding and transcription initiation. Weak promoters and activating factor

52. Molecular Biology Course K4 Transcription process 1.Promoter binding 2.DNA unwinding 3.RNA chain initiation 4.RNA chain elongation 5.RNA chain termination (  factor)

53. 1.Promoter binding K4 Transcription process The searching process is extremely rapidly Closed complex : the initial complex of the polymerase with the base-paired promoter DNA) and –10 region Link

54. The RNA polymerase core enyzme,    ’  has a general non-specific affinity for DNA, which is referred to as loose binding that is fairly stable. The addition of  factor to the core enzyme markedly reduces the holoenzyme affinity for non-specific binding by 20 000-fold, and enhances the holoenzyme binding to correct promoter sites 100 times. Overall,  factor binding dramatically increases the specificity of the holoenzyme for correct promoter-binding site. The role of  factor in promoter binding K4 Transcription process

55. 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

56. It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing and RNA synthesis. Negative supercoiling 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 supercoiling & unwinding K4 Transcription process

57. 3. RNA chain initiation +1 The polymerase initially incorporates the first two nucleotides and forms a phosphodiester bond. K4 Transcription process Starts with a GTP or ATP

58. Abortive initiation The RNA pol. goes through multiple abortive initiations before a successful initiation, which limits the overall rate of transcription The minimum time for promoter clearance is 1-2 seconds (a long event, the synthesis is 40 nt/ sec) The first 9 nt are incorporated without polymerase movement along the DNA. Afterward, there is a significant probability that the chain will be aborted. K4 Transcription process

59. 4. RNA chain elongation K4 Transcription process

60. Promoter clearance: when initiation succeeds, the enzyme releases  factor and forms a ternary complex of polymerase-DNA-nascent RNA, causing the polymerase to progress along the DNA to allow the re-initiation of transcription. K4 Transcription process

61. Transcription bubble: 1.containing ~ 17 bp of unwound DNA region and the 3’-end of the RNA that forms a hybrid helix about 12 bp. 2.moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear. 3.The E. coli polymerase moves at an average rate of ~ 40 nt per sec, depending on the local DNA sequence. K4 Transcription process

62. Transcription bubble

63. 5. RNA chain termination 1.Termination occurs at terminator DNA sequences. 2.The most common stop signal is an RNA hairpin (self-complement structure) commonly GC-rich to favor the structure stability 3. Rho-dependent and Rho-independent Termination.

64. Terminator A specific DNA sequence where the transcription complex dissociate Rho protein (  ) independent terminator contains: (1) self-complementary region that is G-C rich and can form a stem-loop or hairpin secondary structure. GC-rich favouring the base pairing stability and causing the polymerase to pause. (2) a run of adenylates (As) in the template strand that are transcribed into uridylates (Us) at the end of the RNA, resulting in weak RNA- antisense DNA strand binding.

65. A model for  - independent termination of transcription in E. coli. The A-U base-pairing is less stable that favors the dissociation

66. 1. Contains only the self-complementary region 2. Requires  protein for termination   protein binds to specific sites in the single- stranded RNA   protein hydrolyzes ATP and moves along the nascent RNA towards the transcription complex then enables the polymerase to terminate transcription Rho protein (  ) dependent terminator

68. RNA polymerase/transcription and DNA polymerase/replication RNA polDNA pol TemplatedsDNA is better than ssDNA dsDNA Require primerNoYes Initiationpromoterorigin elongation40 nt/ sec900 bp/sec terminatorSynthesized RNATemplate DNA

69. K: supplemental 1 In any chromosome, different genes may use different strands as template (Fig. 25-2).

70. Thanks