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Molecular Biology Course

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  1. Molecular Biology Course Section K: Transcription in prokaryotes

  2. Section K: Transcription in prokaryotes K1 Basic principles of transcription An overview, the process of RNA synthesis ( initiation, elongation, termination) K2 Escherichia coli RNA polymerase Properties, a subunit, b subunit, b’ subunit, sigma (s) factor K3 The E. colis70 promoter Promoter, s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency K4 transcription process. Promoter binding, unwinding, RNA chain initiation, elongation, termination (r factor)

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

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

  5. Key terms defined in this section (Gene VII) +1 Gene X upstream downstream Primary transcript m7Gppp mRNA AAAAAn 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.

  6. 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. Promoteris a region of DNA involved in binding of RNA polymerase to initiate transcription

  7. 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.

  8. K1: Basic principles of transcription 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

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

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

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

  12. K1: Basic principles of transcription Transcription

  13. K1: Basic principles of transcription • RNA synthesis occurs in the 5’3’ 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 &terminator/template

  14. K1: Basic principles of transcription K1-2: The process of RNA synthesis • initiation • elongation • termination

  15. Flowchart of RNA synthesis Back 1, 2

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

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

  18. 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

  19. 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)

  20. Terminator structure

  21. Molecular Biology Course K2 Escherichia coli RNA polymerase • E. coli RNA polymerase • a subunit • b subunit • b’ subunit • sigma (s) factor

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

  23. K2: E. coli RNA polymerase RNA polymerase (NMP)n + NTP  (NMP)n+1 + PPi • Requires no primer for polymerization • Requires DNA for activity and is most active with a double-stranded DNA as template. • 5’  3’ synthesis • Require Mg2+ for RNA synthesis activity • 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.

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

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

  26. K2: E. coli RNA polymerase 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

  27. K2: E. coli RNA polymerase K2-2: a subunit

  28. K2: E. coli RNA polymerase E. coli polymerase: a 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

  29. K2: E. coli RNA polymerase K2-3&4: b and b’ subunit

  30. b is encoded by rpoB gene, and b’ is encoded by rpoC gene . • Make up the catalytic center of the RNA polymerase • 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. • The b 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 b’ also is involved at all stages.

  31. K2: E. coli RNA polymerase • b 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.

  32. K2: E. coli RNA polymerase b’ subunit • Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase • Hyparin (肝素):binds to 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 .

  33. K2: E. coli RNA polymerase K2-5: Sigma (s) factor

  34. Many prokaryotes contain multiple s factors to recognize different promoters. 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 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.

  35. Molecular Biology Course K3: The E. colis70 promoter • Promoter • s70 size • -10 sequence • -35 sequence • transcription start site • promoter efficiency

  36. K3: The E. colis70 promoter K3-1: Promoter • The specific short conserved DNA sequences: • upstream from the transcribed sequence, and thus assigned a negative number (location) • required 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

  37. +1 Promoter Terminator Sense strand DNA Transcribed region Antisense strand Transcription RNA K3: The E. colis70 promoter Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

  38. K3: The E. colis70 promoter K3-2,3&4: s70 promoter

  39. K3: The E. colis70 promoter ---5-8 bp--- G C T A TTGACA -----16-18 bp------- TATAAT -35 sequence -10 sequence +1 • 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

  40. K3: The E. colis70 promoter -10sequence (Pribonow box) • The most conserved sequence in s70 promoters at which DNA unwinding is initiated by RNA Pol. • A 6 bp sequence which is centered at around the –10 position, and is found in the promoters of many different E. coli gene. • The consensus sequence is TATAAT. The first two bases (TA) and the final T are most highly conserved. • This hexamer is separated by between 5 and 8 bp from position +1, and the distance is critical.

  41. K3: The E. colis70 promoter -35 sequence: enhances recognition and interaction with the polymerase s 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

  42. Supplemental material RNA Polymerase Leaves Its FootPrint on a Promoter • 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.

  43. Supplemental material Footprinting

  44. Supplemental material Footprinting

  45. K3: The E. colis70 promoter K3-5: 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)

  46. K3: The E. colis70 promoter The sequences of five E. coli promoters

  47. K3: The E. colis70 promoter K3-6: promoter efficiency There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold .

  48. 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 sequence 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.

  49. Weak promoters and activating factor 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.