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Chapter 31

Chapter 31. The Prokaryotic Transcription Apparatus (pages 1014-1023). Learning objectives: Understand the following Differences between DNA and RNA polymerases E. coli RNA polymerase subunits and their function What sequences make up a prokaryotic promoter

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Chapter 31

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  1. Chapter 31 The Prokaryotic Transcription Apparatus (pages 1014-1023) • Learning objectives: Understand the following • Differences between DNA and RNA polymerases • E. coli RNA polymerase subunits and their function • What sequences make up a prokaryotic promoter • The steps of transcription initiation and elongation • The two different mechanisms of transcription termination

  2. Recombination Mutation/Repair Flow of genetic information - overview

  3. 3 Different Classes of RNA 1) rRNA (ribosomal) • large (long) RNA molecules • structural and functional components of ribosomes • few types, highly abundant 2) mRNA (messenger) • typically small (short) • encode proteins • multiple types, not abundant 3) tRNA (transfer) and small ribosomal RNAs • very small • Important in translation Not all genes encode proteins!

  4. Different Types of RNA Polymerase • In Bacteria(simple system) • - all three classes are transcribed by the • same RNA polymerase (RNAP for short) • (note that we don’t include primase in our discussion of RNA polymerases) • In Eukaryotes(complex system) • - each class is transcribed by a different • RNA Polymerase • RNAP I - rRNAs • RNAP II - mRNAs • RNAP III - tRNAs & small ribosomal RNAs

  5. All RNA polymerases require: 1) DNA template: one strand is copied 2) substrate NTPs (GTP, CTP, UTP, ATP) 3) divalent cation (Mg2+) Differences Between DNAP and RNAP 1) RNAP can initiate transcription de novo (i.e. RNAP doesn’t need a primer!) 2) RNAP has lower levels of proofreading activity (error rate is 1 in 104 or 105 ntds added) 3) RNAP incorporates NTPs instead of dNTPs 4)RNAP incorporates UTP instead of dTTP

  6. Similarities and differences between DNA and RNA RNA DNA • Similar strand structure • Can define a 5’ and 3’ end • 2’ hydroxyl in RNA • (causes stability differences) • Uracil in RNA not in DNA

  7. DNA strand RNA strand Subsequent hydrolysis of PPi drives the reaction forward OH OH RNA synthesis is in the 5’ to 3’ direction RNA has polarity (5’ phosphate, 3’ hydroxyl)

  8. Some nomenclature conventions (sense strand) (coding strand) RNAP (anti-sense strand) (non-coding strand)

  9. Transcription Initiation Site +1 Direction of transcription 5’ 3’ 3’ 5’ -5 -2 -3 -1 -4 +2 +3 +4 +5 +6 Template strand Other important nomenclature conventions “Upstream” “Downstream” There is no “zero”

  10. Bacterial (Prokaryotic) Transcription • Promoters • - DNA sequences that guide RNAP to the beginning • of a gene (transcription initiation site) • RNA Polymerase (RNAP) • - subunit structure/function • Reaction (ordered series of steps) • 1) initiation • 2) elongation • 3) termination

  11. Mapping Promoters • - DNA sequences that guide RNAP to the start of a gene (transcription initiation site)

  12. RNAP binds a region of DNA from -40 to +20 The sequence of the non-template strand is shown -10 region TTGACA…16-19 bp... TATAAT “-35” spacer “-10”

  13. Important Promoter Features (tested by mutations) • the closer the match to the consensus the stronger the • promoter (-10 and -35 boxes) • the absolute sequence of the spacer region (between • the -10 and -35 boxes) is not important • the length of the spacer sequence IS important: • TTGACA - spacer (16 to 19 base pairs) - TATAAT • spacers that are longer or shorter than the consensus • length make weak promoters

  14. Properties of Promoters • Promoters typically consist of 40 bp region on the 5'-side of the transcription start site • Two consensus sequence elements: • The "-35 region", with consensus TTGACA • The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding - why?

  15. RNA polymerase has many functions • Scan DNA and identify promoters • Bind to promoters • Initiate transcription • Elongate the RNA chain • Terminate transcription • Be responsive to regulatory proteins (activators and repressors) Thus, RNAP is a multisubunit enzyme

  16. Transcription in Prokaryotes • In E.coli, RNA polymerase is 465 kD complex, with 2 , 1 , 1 ', 1  (holoenzyme) • Core enzyme is 2 , 1 , 1 ’ (can transcribe but it can’t find promoters) •  recognizes promoter sequences on DNA • ' binds DNA;  binds NTPs and interacts with  •  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins

  17. a a2 a2b a2bb’ = core enzyme aI aII b b’ s70 s32 s60 vegetative (principal s) heat shock (for emergencies) nitrogen starvation (for emergencies) The assembly pathway of the core enzyme (the w subunit makes this more efficient) CORE ENZYME Sequence-independent, nonspecific transcription initiation + SIGMA SUBUNIT interchangeable, promoter recognition

  18. aI aII RNAP HOLOENZYME -s70 Promoter-specific transcription initiation b b’ s70 • In the Holoenzyme: • ' binds DNA •  binds NTPs •  and  ' together make up the active site •  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. • s recognizes promoter sequences on DNA

  19. RNAP core structure from T. aquaticus. • RNAP is a “crab claw” shape with a wide channel to bind DNA and RNA. • The active site is at the base of the two “pincers”. The pincers are flexible and allow conformation changes during transcription. PNAS January 30, 2001 volume 98 page 892-897

  20. Binding of polymerase to Template DNA • Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter • Sigma subunit recognizes promoter sequence • RNA polymerase holoenzyme and promoter form "closed promoter complex" (DNA not unwound) - Kd = 10-6 to 10-9 M • Polymerase unwinds about 12 base pairs to form "open promoter complex" - Kd = 10-14 M

  21. Initiation of Polymerization • RNA polymerase has two binding sites for NTPs • Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end) • Elongation site binds the second incoming NTP • 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) • When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation"

  22. RNAP bound -40 to +20 Closed complex formation RNAP unwinds from -10 to +2 Open complex formation Requires high purine [NTP] Binding of 1st NTP Requires lower [NTPs] Addition of next NTPs After RNA chain is 6-10 NTPs long Dissociation of sigma Finding and binding the promoter

  23. Chain Elongation Core polymerase - no sigma • Polymerase is pretty accurate - only about 1 error in 10,000 bases (not as accurate as DNAP III) • Even this error rate is OK, since many transcripts are made from each gene • Elongation rate is 20-50 bases per second - slower in G/C-rich regions and faster elsewhere • Topoisomerases precede and follow polymerase to relieve supercoiling

  24. Interactions between nucleic acids and the core enzyme keep RNAP processive Downstream DNA Upstream DNA

  25. A high-resolution view of RNA polymerase Elongating core Holo Open complex From: Cell, Vol 109, 417-420, May 2002

  26. Chain Termination Two mechanisms 1) Rho - the termination factor protein • rho is an ATP-dependent helicase • it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain

  27. Rho-Dependent Transcription Termination (depends on a protein AND a DNA sequence) G/C -rich site RNAP slows down Rho helicase catches up Elongating complex is disrupted

  28. Chain Termination Two mechanisms 2) Rho-Independent - termination sites in DNA • inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript • 6-8 A’s in DNA coding for U’s in transcript

  29. Rho-independent transcription termination (depends on DNA sequence - NOT a protein factor) Stem-loop structure

  30. Rho-independent transcription termination • RNAP pauses when it reaches a termination site. • The pause may give the hairpin structure time to fold • The fold disrupts important interactions between the RNAP and its RNA product • The U-rich RNA can dissociate from the template • The complex is now disrupted and elongation is terminated

  31. We have now finished Chapter 31 Section 1 For next class please read: Chapter 31 section 2 Pages 1024-1028

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