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. Flow of genetic information - overview Recombination Mutation/Repair

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. 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 - rRNAsRNAP I - rRNAs RNAP II - mRNAsRNAP II - mRNAs RNAP III - tRNAs & small ribosomal RNAsRNAP III - tRNAs & small ribosomal RNAs Different Types of RNA Polymerase

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 10 4 or 10 5 ntds added) 3) RNAP incorporates NTPs instead of dNTPs 4)RNAP incorporates UTP instead of dTTP

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

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

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

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

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

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

12. -10 region RNAP binds a region of DNA from -40 to +20 The sequence of the non-template strand is shown 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.        ’ = core enzyme   ’’  CORE ENZYME Sequence-independent, nonspecific transcription initiation + vegetative (principal  )   heat shock (for emergencies)   nitrogen starvation (for emergencies)   SIGMA SUBUNIT interchangeable, promoter recognition The assembly pathway of the core enzyme (the  subunit makes this more efficient)

18.   ’’    RNAP HOLOENZYME -  70 Promoter-specific transcription initiation 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. ·  recognizes promoter sequences on DNA

19. RNAP core structure from T. aquaticus. PNAS January 30, 2001 volume 98 page 892-897 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.

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) - K d = 10 -6 to 10 -9 M Polymerase unwinds about 12 base pairs to form "open promoter complex" - K d = 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 PP i ) When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation"

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

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

26. 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 Chain Termination

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. 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 Chain Termination

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. Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company We have now finished Chapter 31 Section 1 For next class please read: Chapter 31 section 2 Pages 1024-1028